Genetic Engineering [Volume 2: Applications, Bioethics, and Biosafety] 9781774912713, 9781774912720, 9781774912676, 9781774912683, 9781003378266, 9781774912690, 9781774912706, 9781003378273

Table of contents :
Cover
Half Title
Title Page
Copyright Page
Dedication
About the Editors
Table of Contents
Contributors
Abbreviations
Acknowledgment
Foreword
Preface
1. Biosafety, Intellectual Property Rights (IPR), and Protection (IPP)
2. Safety and Benefits of Bt and Bt Cotton: Factures, Refute, and Allegations
3. Biohazards of Recombinant DNA Technology
4. Genetic Engineering and Human Welfare
5. Genetically Modified Organisms: Scope and Challenges
6. Genetic Engineering of Horticultural Crops
7. Genetically Engineered Microorganisms
8. Patenting of Living Organisms: Significance, Copyrights, Trade Secrets, and Trademarks
9. Negative Impact of Recombinant DNA Technology on Life
10. Genetic Engineering and Agricultural Sciences
11. Construction of Recombinant DNA
12. Genetically Modified Organisms: Concerns and Biosafety
Index

Citation preview

GENETIC ENGINEERING

Volume 2

Applications, Bioethics, and Biosafety

Genetic Engineering, 2-volume set ISBN: 978-1-77491-271-3 (hbk) ISBN: 978-1-77491-272-0 (pbk) Genetic Engineering, Volume 1: Principles Mechanism, and Expression ISBN: 978-1-77491-267-6 (hbk) ISBN: 978-1-77491-268-3 (pbk) ISBN: 978-1-00337-826-6 (ebk) Genetic Engineering, Volume 2: Applications, Bioethics, and Biosafety ISBN: 978-1-77491-269-0 (hbk) ISBN: 978-1-77491-270-6 (pbk) ISBN: 978-1-00337-827-3 (ebk)

GENETIC ENGINEERING

Volume 2

Applications, Bioethics, and Biosafety

Edited by:

Tariq Ahmad Bhat, PhD

Jameel M. Al-Khayri, PhD

First edition published 2024 Apple Academic Press Inc. 1265 Goldenrod Circle, NE, Palm Bay, FL 32905 USA 760 Laurentian Drive, Unit 19, Burlington, ON L7N 0A4, CANADA

CRC Press 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742 USA 4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN UK

© 2024 by Apple Academic Press, Inc. Apple Academic Press exclusively co-publishes with CRC Press, an imprint of Taylor & Francis Group, LLC Reasonable efforts have been made to publish reliable data and information, but the authors, editors, and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors, editors, and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged, please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. For works that are not available on CCC please contact [email protected] Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. Library and Archives Canada Cataloguing in Publication

CIP data on file with Canada Library and Archives

Library of Congress Cataloging-in-Publication Data

CIP data on file with US Library of Congress

ISBN: 978-1-77491-269-0 (hbk) ISBN: 978-1-77491-270-6 (pbk) ISBN: 978-1-00337-827-3 (ebk)

Dedication

This book is dedicated to:

Ibn Al-Baitar (1197–1248) Arab scientist, botanist, and physician who systematically recorded the discoveries made by Islamic physicians in the middle ages.

About the Editors

Tariq Ahmad Bhat, PhD Lecturer on Botany, Department of Education, Govt. of Jammu and Kashmir, India Tariq Ahmad Bhat, PhD, with 18 years of teaching experience, is a lecturer on botany in the Department of Education, Govt. of Jammu and Kashmir, India, and is engaged in active research of molecular biology, cell biology, mutation breeding, and genetic improvement of legumes and medicinal plants. He has published 10 international books, 90 research papers, review articles, and book chapters. He has participated in 50 conferences, training programs, and workshops. He is one of the founder faculty members of the Chief Minister’s Super 50 NEET Programme in Jammu and Kashmir, India. He is serving as the District Coordinator Anantnag of a prestigious project on medicinal plants under financial assistance of the National Medicinal Plants Board (NMPB), New Delhi (Ministry of AYUSH), India. The Government of India conferred on him the Best innovative Science Teacher Award 2014 in recognition of his meritorious services. Dr. Bhat has received his MSc and PhD from AMU, Aligarh India. Jameel M. Al-Khayri, PhD Professor of Plant Biotechnology, Department of Agricultural Biotechnology, College of Agriculture and Food Sciences, King Faisal University, Saudi Arabia Jameel M. Al-Khayri, PhD, is Professor of Plant Biotechnology at the Department of Agricultural Biotechnology, College of Agriculture and Food Sciences, King Faisal University, Saudi Arabia.

viii About the Authors

He has dedicated his research efforts on date palm biotechnology for the last three decades. He has published over 60 research articles and reviews in international journals in addition to 30 book chapters. Dr. Al-Khayri is editor of several special issues of international journals on date palm, biotechnology, and sustainable agriculture under abiotic and biotic stress. He is editor of 15 Springer reference books, including Date Palm Biotechnology, Date Palm Genetic Resources and Utilization (2 volumes), Date Palm Biotechnology Protocols (2 volumes), and Advances in Plant Breeding Strategies (9 volumes). He is a member of the editorial board and reviewers’ panels of several international journals. He has participated in the organizing and scientific committees of international scientific conferences and contributed over 50 research presentations. In addition to teaching, graduate students advising, and conducting funded research projects, he has held administrative posts as Assistant Director of the Date Palm Research Center, Head of Department of Plant Biotechnology, and Vice Dean for Development and Quality Assurance. Dr. Al-Khayri is an active member of the International Society for Horticultural Science and the Society for In Vitro Biology and serves as the National Correspondent of the International Association of Plant Tissue Culture and Biotechnology. He served as a member of Majlis Ash-Shura (Saudi Arabia Legislative Council) Fifth Session. Currently, he maintains an active research program on date palm focusing on genetic transformation, secondary metabolites, and in vitro mutagenesis to enhance tolerance to abiotic and biotic stress. He is interested in the role of biotechnology in enhancing food security and the impact of global climate change on agriculture. Dr. Al-Khayri earned a BS in Biology from the University of Toledo and an MS in Agronomy and PhD in Plant Science from the University of Arkansas, USA.

Contents

Contributors......................................................................................................... xi

Abbreviations ..................................................................................................... xiii

Acknowledgment ............................................................................................... xvii

Foreword by Abdul Rauf Shakoori..................................................................... xix

Preface ............................................................................................................... xxi

1.

Biosafety, Intellectual Property Rights (IPR), and Protection (IPP) ...... 1

Muhammad Ishtiaq, Muhammad Waqas Mazhar, Mehwish Maqbool, Muhammad Ajaib, Tanveer Hussain, Mahnoor Muzamil, and Mubashir Mazhar

2. Safety and Benefits of Bt and Bt Cotton: Factures, Refute, and Allegations.............................................................................. 23

Surendra Singh Shekhawat, Irsad, and Safdar Kaiser Hasmi

3.

Biohazards of Recombinant DNA Technology ........................................ 53

Johra Khan

4.

Genetic Engineering and Human Welfare............................................... 73

Mohd. Suliman Dar and Shugufta Rasheed

5. Genetically Modified Organisms: Scope and Challenges....................... 89

Muhammad Ishtiaq, Mubashir Mazhar, Mehwish Maqbool, and Muhammad Waqas

6.

Genetic Engineering of Horticultural Crops......................................... 107

Muhammad Ishtiaq, Mubashir Mazhar, Mehwish Maqbool, and Mahnoor Muzamil

7.

Genetically Engineered Microorganisms............................................... 125

Praful Upendra Saha, Ahmad Ali, and Johra Khan

8. Patenting of Living Organisms: Significance, Copyrights, Trade Secrets, and Trademarks.............................................................. 159

Muhammad Sajjad Iqbal and Faiza Nasir

9.

Negative Impact of Recombinant DNA Technology on Life ................ 183

Twinkle Dixit, Namrata Dutta, and Arpit Shukla

x Contents

10. Genetic Engineering and Agricultural Sciences.................................... 205

Muhammad Ishtiaq, Muhammad Waqas Mazhar, and Mehwish Maqbool

11. Construction of Recombinant DNA ....................................................... 221

Mohammad Amin Lone and Anzar A. Shah

12. Genetically Modified Organisms: Concerns and Biosafety ................. 245

Muhammad Ishtiaq, Mubashir Mazhar, Mehwish Maqbool, and

Muhammad Waqas Mazhar

Index ................................................................................................................. 259

Contributors

Muhammad Ajaib

Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur, Azad Jammu and Kashmir (AJK), Pakistan

Ahmad Ali

Department of Life Sciences, University of Mumbai, Vidyanagari, Santacruz, Mumbai, Maharashtra, India

Mohd. Suliman Dar

Department of Botany Government Degree College, Anantnag, Jammu and Kashmir, India

Twinkle Dixit

Department of Microbiology and Biotechnology, Gujarat University, Ahmedabad, Gujarat, India

Namrata Dutta

Department of Microbiology, Swarrnim Startup and Innovation University, Adalaj Kalol Highway, Gandhinagar, Gujarat, India

Safdar Kaiser Hasmi

Department of Plant Protection, Aligarh Muslim University, Aligarh, Uttar Pradesh, India

Tanveer Hussain

Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur, Azad Jammu and Kashmir (AJK), Pakistan

Muhammad Sajjad Iqbal

Biodiversity Informatics, Genomics, and Post-Harvest Biology, Department of Botany, University of Gujrat, Gujrat, Pakistan

Irsad

Department of Plant Protection, Aligarh Muslim University, Aligarh, Uttar Pradesh, India

Muhammad Ishtiaq

Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur, Azad Jammu and Kashmir (AJK), Pakistan

Johra Khan

Department of Medical Laboratory Sciences, College of Applied Medical Sciences; Majmaah University, Majmaah, Saudi Arabia

Mohammad Amin Lone

Department of Zoology, Government Degree College (GDC), Uri, Jammu and Kashmir, India

Mehwish Maqbool

Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur, Azad Jammu and Kashmir (AJK), Pakistan

xii

Contributors

Mubashir Mazhar

Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur, Azad Jammu and Kashmir (AJK), Pakistan

Muhammad Waqas Mazhar

Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur, Azad Jammu and Kashmir (AJK), Pakistan

Mahnoor Muzamil

Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur, Azad Jammu and Kashmir (AJK), Pakistan

Faiza Nasir

Biodiversity Informatics, Genomics, and Post-Harvest Biology, Department of Botany, University of Gujrat, Gujrat, Pakistan

Shugufta Rasheed

CBT, Department of Botany, University of Kashmir, Srinagar, Jammu and Kashmir, India

Praful Upendra Saha

Department of Life Sciences, University of Mumbai, Vidyanagari, Santacruz, Mumbai, Maharashtra, India

Anzar A. Shah

Department of Zoology, Government Degree College (GDC), Uri, Jammu and Kashmir, India

Surendra Singh Shekhawat

Department of Plant Protection, Aligarh Muslim University, Aligarh, Uttar Pradesh, India

Arpit Shukla

Department of Biological Sciences and Biotechnology, Institute of Advanced Research, University of Innovation, Koba Institutional Area, Gandhinagar, Gujarat, India

Muhammad Waqas

Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur, Azad Jammu and Kashmir (AJK), Pakistan

Abbreviations

ACC

1-aminocyclopropane-1-carboxylate

AMV

avian myeloblastosis virus

APHIS

animal and plant health inspection services

BCH

biosafety clearing house

BST

bovine somatotrophin

Bt

Bacillus thuringiensis

BTV

bluetongue virus

CaMV

cauliflower mosaic virus

CaMV35S

cauliflower mosaic virus 35S

CB

cost: benefit

CBD

convention on biological diversity

cDNA

complementary DNA/copy DNA

CERA

Center for Environmental Risk Assessment

CITES

convention on international trade in endangered species

CNIPA

China National Intellectual Property Administration

CpTI

cowpea trypsin inhibitor

CRISPR

clustered randomly interspaced short palindromic repeats

DNA

deoxyribonucleic acid

DUS

distinctive, uniform, stable

E. coli

Escherichia coli

EIQ

ecosystem impact quotient

EPA

Environmental Protection Agency

EPC

European Patent Convention

EPO

European Patent Office

FAO

Food and Agriculture Organization

xiv

Abbreviations

FDA

Food and Drug Administration

FSE

farm scale evaluations

GCSF

granulocyte colony-stimulating factors

GD

genetic diversity

GEAC

genetic engineering approval committee

GEMs

genetically engineered microorganisms

GEOs

genetically engineered organisms

GEPs

genetic engineering principles

GET

genetic engineering technology

GGPP

geranylgeranyl diphosphate

GM

genetically modified

GmCSF

granulocyte-macrophage colony-stimulating factors

GMM

genetically modified microorganisms

GMO

genetically modified organism

GNA

Galanthus nivalis

HIV

human immunodeficiency virus

IARC

International Agency for Research on Cancer

ICGEB

International Center for Genetic Engineering and Biotechnology

IDA

International Depositary Authorities

IPO

Indian Patent Office

IPO

Intellectual Property Organization

IPP

intellectual property protection

IPPC

International Plant Protection Convention

IPR

intellectual property rights

JH

juvenile hormone

JPO

Japan Patent Office

KIPO

Korean Intellectual Property Office

lncRNA

long non-coding RNA

Abbreviations

xv

MAHYCO

Maharashtra Hybrid Seed Company

MMTV

mouse mammary tumor virus

MuLV

murine leukemia virus

N. crassa

Neurosporra crasa

NHEJ

nonhomologous end joining

NIH

National Institute of Health

OECD

Organization for Economic Co-operation and Development

PBW

pink bollworm

PCR

polymerase chain reaction

PCT

patent cooperation treaty

PICPB

Paris International Convention for Protection of Birds

PPA

plant patent Act

rDNA

recombinant DNA

RDT

recombinant DNA technology

REs

restriction enzymes

RFLP

restriction fragment length polymorphism

RGA

rapid generations advances

RSV

Rous sarcoma virus

SAR

systemic acquired resistance

SARS-CoV-2

severe acute respiratory syndrome coronavirus

S-GEMS

suicidal genetically engineered microorganisms

SIP

secure isotope probing

siRNAs

small interfering RNAs

SSNs

site-specific nucleases

TALE

TAL effector

TALENs

transcription activator-like effector nuclease

TMR

trademark register

UK

United Kingdom

xvi

Abbreviations

UNO

United Nations Organization

UPOV

Union for the Protection of New Varieties

USDA

United States Department of Agriculture

USPTO

US Patent and Trademark Office

UTSA

Uniform Trade Secrets Act

WHC

Western Hemisphere Convention

WHO

World Health Organization

WIPO

World Intellectual Property Organization

WTO

World Trade Organization

ZF

zinc fingers

ZFNs

zinc finger nucleases

Acknowledgment

The editors extend their appreciation to the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia for supporting this work through Project No. GRANT3101.

Foreword

Genetic engineering is the process of using recombinant DNA technology (RDT) to change/alter the genetic makeup of an organism. This technology has applications in medicine, research, industry, and agriculture and can be used on a wide range of plants, animals, and microorganisms. A variety of drugs and hormones for medical use have been produced by genetic engineering. Some of the earlier medicines manufactured by this technology were insulin, somatostatin, Interferon, plasminogen activator, and urokinase. In addition, modified plasmids or viruses have been used as messengers to deliver genetic material to the body’s cells, resulting in the production of substances that should correct the illness. Sometimes cells are genetically altered inside the body; other times, scientists modify them in the laboratory and return them to the patient’s body. Since the 1990s, gene therapy has been used in clinical trials to treat diseases and conditions such as AIDS, cystic fibrosis, cancer, and high cholesterol. George Kohler and Cesar Milstein produced identical antibodies by hybridoma technology. Recombinant DNA technology has been extensively exploited to alter the genotype of crop plants to make them more productive, nutritious, rich in proteins, disease resistant, and less fertilizer consuming. In combination with tissue culture techniques, genetic engineering techniques have been used to produce high-yielding cereals, pulses, and vegetable crops. Scientists have developed transgenic potato, tobacco, cotton, corn, strawberry, and rape seeds that are resistant to insect pests and certain weedicides. Genetically designed bacteria are put into use for generating industrial chemicals. A variety of organic chemicals can be synthesized at a large scale with the help of genetically engineered microorganisms (GEMs). Glucose can be synthesized from sucrose with the help of enzymes obtained from genetically modified (GM) organisms.

xx

Foreword

Recombinant DNA technology can also be used to monitor the degradation of garbage, petroleum products, naphthalene, and other industrial wastes. Volume II of the book Genetic Engineering covers all the important topics concerning applications of recombinant DNA technology in the domain of human welfare, agriculture, and horticulture. The scope and challenges of genetically modified organisms (GMOs) (microorganisms as well as plants) have been given special treatment in the book. Issues like negative implications of genetically modified organisms and products, biosafety, intellectual property rights (IPRs), patenting, and copyrights are the highlights of the book, which are highly informative and thoughtprovoking aspects of genetic engineering. I applaud the editor, Dr. Tariq Ahmad Bhat, as well as the book chapter contributors for successfully bringing together this volume. —Abdul Rauf Shakoori Distinguished National Professor,

Professor Emeritus,

University of the Punjab, Lahore, Pakistan

Preface

The advent of biotechnology has forever changed human perception of living entities. Genetic engineering enables the precise control of the genetic composition and gene expression of organisms directed toward the advantage of human well-being. Innovative applications have emerged in environmental sustainability, food and nutritional security, and medicinal advancement. The utilization of this powerful technology solicits contrasting opinions among scientists, politicians, and the public in relation to biosafety and bioethics. This necessitated the engagement in research aimed at understanding the safety of genetic engineering products and prompted the development of national and international policies. This book addresses these aspects in two volumes: Volume 1, Genetic Engineering: Principles, Mechanism, and Expression, and Volume 2, Genetic Engineering: Applications, Bioethics, and Biosafety. Volume 2 consists of 12 chapters dealing with various appreciations and challenges of genetic engineering and issues related to bioethics and biosafety. The chapters of this volume are biosafety, intellectual property rights (IPR) and protection (IPP), safety and benefits of Bt and Bt cotton: factures, refutations, and allegations, biohazards of recombinant DNA technology (RDT), genetic engineering and human welfare, genetically modified (GM) organisms: scope and challenges, genetic engineering of horticultural crops, genetically engineered microorganisms (GEMs), patenting of living organisms (copyrights, trade secrets and trademarks), the negative impact of recombinant DNA technology on life, genetic engineering and agricultural sciences, and construction of recombinant DNA (rDNA). This book is a valuable asset to upper-undergraduate and postgraduate students, teachers, and researchers interested in cell biology, genetics, molecular genetics, biochemistry, biotechnology, botany, zoology, and agriculture sciences. The chapters are contributed by experts in their fields, presenting recent contemporary developments in genetic engineering research supported with colored illustrations, tables, and recent references. We are thankful to all the authors across the globe who contributed their research output in the form of book chapters to make our project a

xxii

Preface

successful endeavor. We wish to present our gratitude to the contributing authors for their generous cooperation and to the Apple Academic Press (APP)/CRC Press/Taylor and Francis Group for giving us the opportunity to publish this work. —Tariq Ahmad Bhat, PhD Jammu and Kashmir, India Jameel M. Al-Khayri, PhD Al-Ahsa, Saudi Arabia

CHAPTER 1

Biosafety, Intellectual Property Rights (IPR), and Protection (IPP) MUHAMMAD ISHTIAQ, MUHAMMAD WAQAS MAZHAR, MEHWISH MAQBOOL, MUHAMMAD AJAIB, TANVEER HUSSAIN, MAHNOOR MUZAMIL, and MUBASHIR MAZHAR

Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur, Azad Jammu and Kashmir (AJK), Pakistan

ABSTRACT Plants and their different products obtained through biotechnological approaches are inevitable for life necessities. The advent of different types of varieties, cultivars, or the production of other hormones on a commercial level by applying novel techniques do require the protection of property rights as well as biosafety of procedures keeping humanity secure and safe. Biosafety is the most pivotal; otherwise, if all biotechnological processes are left open may cause loss at the individual level or massive catastrophe due to sudden bio-war through terrorist activities. The safety, security, and proper replication without any accidental or incidental mutation is a prerequisite of the biosafety of biotechnological products. Intellectual property rights (IPRs) and intellectual property protection (IPP) are the next two phases of the safety and security of any biotechnological invention and discoveries. The IPR and IPP provide a fundamental right of protection of novel ideas, neo procedure, products, and then their commercial propagation without any fear of piracy loss and also unnecessary indulging of persons or/and corporates in litigations. So, it is clear Genetic Engineering, Volume 2: Applications, Bioethics, and Biosafety. Tariq Ahmad Bhat & Jameel M. Al-Khayri (Eds.) © 2023 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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Genetic Engineering, Volume 2

from the above that patent registration, copyright protection, and licensing of some procedures are always protected and safe under the umbrella of IPR and IPP, complementing the theme and spirit of biosafety of biological techniques all around the globe for betterment and welfare of humanity as general rule and principle. The safe and food security, in conjunction with other life necessities and accessories of life is only secured when all biotechnological and novel ideas, inventions, and products are properly documented and regulated, which is being fulfilled in letter and spirit by biosafety rules, IPR, and IPP promulgated in each country and generally protected by world international protection organization (WIPO) and other sub-bodies of UNO. 1.1 BIOSAFETY: THE NEED FOR FUTURE Broadly biological safety or biosafety can be defined as the use of knowledge, skill, and preventive measures to protect the earth and its resources from biohazards. A biohazard is any substance that can cause irreversible damage to both the organic and physical environment surrounding humanity, including human beings themselves. There is a delicate balance between nature and human being; however, the anthropogenic activities and fast progress in the modern world of science and technology has led to disturbance of the biological integrity and strict biosafety measures are the need of the hour (Gupta, 2000). The world of science and technology has been ever expanding and the technology intervention into the lives of the people is trending in the modern era. Especially the field of biotechnology is flourishing fast and has enabled the researchers to modify genome by manipulating the foreign genes into the genetic makeup to increase the yield quantity and quality and to maximize the benefits from the animals. The genetic modification leads to the formation of the genetically modified organisms (GMOs). The behavior of GMOs with the natural world is the subject of biosafety as the GMOs intervention in the natural world has led to serious ecological challenges related to environmental integrity and human health (Anuradha, 2005; Navneet, 2014, 2018). The genes given to the crops for insect and pest resistance have saved the crops from insects on many occasions but don’t forget that these insects were also important candidates in pollination. GMOs-mediated environmental safety risks are the subject of researchers nowadays as human intervention in the genome

Biosafety, Intellectual Property Rights (IPR), and Protection (IPP)

3

has led to deterioration of the delicate balance between nature and man. Serious debate on the GMOs intervention in natural ecosystem should be the part of current time and strict biosafety measures are the need of the hour to save the humanity from the biohazardous products (Gupta, 2000; Isaac, 2002; Nestmann et al., 2002). There are unknown risks to the nature and its resources due to release of transgenic products and biological integrity is on the verge of danger. Analysis of the risk is a requirement of the time and capacity building to maximize the benefit of biotechnology and from GMOs is important (Isaac, 2002; Anuradha, 2005). So, the biosafety is the protection of natural resources of the earth from the harms of fluctuations in the world of technology and the aim of biosafety is to maximize the benefit from the biotechnological products with a little loss to the nature and its resources (Gupta, 2000; Nestmann et al., 2002; Lianchawii, 2005). 1.2 RISK MANAGEMENT AND ANALYSIS Release of the genetically altered organisms in the environment carries safety risks and challenges that organism poses to the nature. The risk can be defined as the prediction of the hazard due to introduction of the genetically altered organism or the biologically hazardous organism in the environment. Risk can be predicted and calculated, and its assessment involves a series of steps in which the nature of the hazard is defined on the basis of predicted consequences. The following groups of the risks are generally encountered due to induction of transgenic variety or GMOs into the nature. 1. Risk Group 1 (RG-1): GMOs or the biologically hazardous agents categorized in this group are not known to cause any disease in adult with proper homeostasis, e.g., E. coli. 2. Risk Group 2 (RG-2): GMOs or the biologically hazardous agents categorized in this group are known to cause diseases which are not serious and for which the proper medication is available generally, e.g., Hepatitis B Virus 3. Risk Group 3 (RG-3): GMOs or the biologically hazardous agents categorized in this group are known to cause diseases which are serious and may result in the death; however, the medication

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Genetic Engineering, Volume 2

or vaccination treatment is available for these diseases, e.g., Rhabdovirus 4. Risk Group 4 (RG-4): GMOs or the biologically hazardous agents categorized in this group are known to cause diseases for which there are no medications or vaccines available thus resulting in the highest risk, e.g., Arena Virus

1.3 FOUR LEVELS OF BIOSAFETY Following biosafety levels of genetic engineering are generally recognized against the above-mentioned risk parameters and commonly employed as shown in Figure 1.1: 1. Biosafety Level 1 (BSL-1): This level of biosafety involves the management of the risks associated with RG-1. The hazards classified under the RG-1 demand no particular use of containment or apparatus regarding their control and eradication. 2. Biosafety Level 2 (BSL-2): This level of biosafety involves the management of the risks associated with RG-2. The hazards classified under the RG-2 demand safety and precautionary measures regarding their handling like autoclaves, lab coats, gloves, etc. 3. Biosafety Level 3 (BSL-3): This level of biosafety involves the management of the risks associated with RG-3. The hazards classified under the RG-3 demand a higher level of safety and training for their administration and handling. The laboratories for handling such risk parameters should be purpose built with proper ventilation and medical guidelines. 4. Biosafety Level 4 (BSL-4): This level of biosafety involves the management of the risks associated with RG-1. The hazards classified under the RG-4 demand the highest level of safety and strict precautionary measures including Hazmat suit and self-contained oxygen supply. There should be all apparatuses to destroy the hazards and risks in the working places like UV light rooms, etc.

Biosafety, Intellectual Property Rights (IPR), and Protection (IPP)

5

FIGURE 1.1 Schematic sketch of steps involved in the risk assessment due to induction of biologically hazardous risk group in a natural ecosystem.

1.4 PROTECTION MEASURES STEPS TO COMBAT BIOSAFETY RISKS The following steps are important and recommended in this regard to coping with biosafety risks: i. Hazard identification is the first step that involves the recognition of the nature of hazard associated with entry of a potentially hazardous biological agent in the natural ecosystem. ii. Understanding the adverse implications related to the induction of hazardous material in the natural ecosystem keeping in view the big picture of risk it may pose to the nature, i.e., Hazard analysis.

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Genetic Engineering, Volume 2

iii. The third step in risk assessment of a hazard is called exposure assessment. It is the evaluation of the probable results on human and animal health due to induction of the risk in a natural ecosystem. iv. Finally, risk evaluation is done to understand whether the risk is manageable or not. It also demonstrates the strategy to mitigate the risks associated with the hazard.

The safe induction of a GMO in the natural environment follows the set of rules designed under the Cartagena Protocol which is narrated in the following paragraph. 1.5 CARTAGENA PROTOCOL: THE FIRST INTERNATIONAL PROTOCOL ON BIOSAFETY The overall risks associated with the induction of GMOs and the potential threats have been a major concern in the scientific community across the globe. There was an urgent need of a set of legislations to be followed across the world for the biosafety from the GMOs induced risks. On January 29, 2000, an international set of rules was put forward to ensure the safe manipulation of the biotechnological products in the earth s natural ecosystem and was named as Cartagena Protocol. The United Nations convention on biological diversity (CBD) also chartered the Cartagena protocol to ensure the biosafety-related issues regarding the transboundary movement of organisms across the globe (Gupta, 2000). 1.6 SALIENT FEATURES OF CARTAGENA PROTOCOL 1. The protocol lays emphasis on the protection of biodiversity due to possible risks associated with the induction of GMOs in the natural ecosystems. 2. All international administrating bodies are directed to ban the GMOs, if found hazardous to the human health and the environment. 3. The induction of GMOs in the natural ecosystem having last biodiversity is banned. 4. All countries should also have a domestic framework of laws regarding the safe handling of the GMOs.

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5. The protocol mandates that an Advanced Informed Agreement should be signed before the cross-border movement of a particular GMO. The overall handling risks should be alarmed to the importer of the GMO so that importer can decide whether to accept or reject the biotechnological product. 6. The biosafety clearing house (BCH) is a set under Cartagena Protocol which helps in the transfer of knowledge about the technical and ecological issues related to GMOs. BCH helps the member countries for the safe implementation of protocol related terms. 7. The protocol addresses the need for proper documentation regarding cross border export of a particular GMO. The protocol says that the export document should contain the legal, technical, and scientific approach for handling a particular GMO. The document should contain the information about the GMO, its traits, and guidelines regarding its safe handling. 8. The knowledge about biosafety and safe handling of GMOs should be imparted to each individual. The protocol mentions the need of public awareness and mutual cooperation for the safe handling of genetically modified (GM) commodities. There should be active participation of the public in the GMOs related handling and legal issues regarding biosafety should be implemented. 9. The Cartagena protocol proposes a simplified system for the use of agricultural products transformed through biotechnology or GM food. The protocol addresses the use of BCH as a platform to inform member nations about the direct use of a GMOs within 15 days. The protocol addresses on the need of capacity building for addressing the GMOs related implications. 1.7 CAPACITY BUILDING FOR PROTECTION The Cartagena protocol addresses all member nations to build their skills for application of the protocol in terms of mutual cooperation and human resource management for the safe negotiation of the GMOs and their handling. Following are some key features of the capacity building to ensure the biosafety and protection: • Risk assessment, risk management, detection, and monitoring of GMOs;

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• Institution building including labs and equipment for testing GMOs; • Scientific, technical, and institutional collaboration; • Human resource development including training in scientific skills; • Facilities and methods for inspection of GMOs; • Awareness, education, and participation; • Information sharing and data management.

1.8 ORGANIZATIONAL SETUPS AND DATABASES ON BIOSAFETY AND PROTECTION International Plant Protection Convention (IPPC) IPPC is a treaty that is concerned with the protection of the plants from pest-related damages as certain GMOs can affect the plants by acting as pests. IPPC addresses the nations across the globe to protect the plants against such a hazardous GMO. 1.9 THE CODEX ALIMENTARIUS COMMISSION This commission is primarily concerned with the public health and agricultural products (Scoones, 2006). The food derived from the biotechnological methods is assessed for its safety for the public. The food based on genetic modification is labeled and processed for the public benefit under the Codex Alimentarius Commission. 1.10 CONVENTION ON BIOLOGICAL DIVERSITY (CBD) In 1992, convention on biological diversity (CBD) platform was established to emphasize the need for the protection of biodiversity (Bilderbeek, 1992). CBD addresses the need to conserve the biological diversity from the threats induced by GMOs. Member nations are advised to take strict precautions and risk assessment associated with the entry of GMOs in biodiverse regions.

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1.11 WORLD TRADE ORGANIZATION (WTO) The World Trade Organization (WTO) deals with the legislations about the global trade. WTO aims to ensure the safe trade between the member countries. The GMOs and genetically transformed products are traded in a safe manner following the set of rules given by the WTO (Pray et al., 2005). 1.12 FOOD AND AGRICULTURE ORGANIZATION (FAO) OF THE UNITED NATIONS An international portal (http://www.ipfsaph.com) for the biosafety regarding GM foods and crops works under the united nation organization about food and agriculture (FAO). FAO ensures the safety of food and agriculture among the member nations and lays emphasis on utilization of the modern agricultural set ups to meet global food demand (Horbulyk, 1993; Scoones, 2006). 1.13 INTERNATIONAL CENTER FOR GENETIC ENGINEERING AND BIOTECHNOLOGY (ICGEB) International Center for Genetic Engineering and Biotechnology (ICGEB) is an international center (http://rasm.icgeb.org) which maintains a database of the Governments decision regarding the risk management related with induction of a GMO in a natural ecosystem (Singh et al., 2004). The system provides documents related to analysis of risk associated with a particular GMO and thus helps to guide the safe handling of GMOs. 1.14 ORGANIZATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT (OECD) Organization for Economic Co-Operation and Development (OECD) ensures the mutual cooperation between various nations to elaborate the knowledge about biosafety and environmental concerns regarding the GNO induction. It particularly focuses on the food security and safety

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and provides a database based on the information regarding global climate change and GM food items and commodities. 1.15 CENTER FOR ENVIRONMENTAL RISK ASSESSMENT (CERA) Center for environmental risk assessment (CERA) was established in March 2009 (http://cera-gmc.org) and it provides information regarding the benefits and demerits of GM crops. It addresses the biosafety issues and sustainable agricultural developments according to the environment (Scoones, 2006). 1.16 GENETICALLY ENGINEERED PRODUCTS AND RECOMBINANT DNA TECHNOLOGY (RDT) Modern era of biotechnology has enabled the researchers across the globe to recombine the DNA from two different resources. In this regard, various methods and techniques have been developed since last two decades. The knowledge of genetic engineering deals with the manipulation of nucleic acid for human welfare and the recombinant DNA technology (RDT) is the technology which is used to modify the genetic makeup of an organism thus leading to the formation of a GMO as stated by Navneet (2014, 2018). RDT involves an overall process in which genes with desired nucleotide sequence are cut and manipulated to achieve a particular eugenic aim. Following are some aims of the RDT: • Understanding the nature of genes and gene products; • Altering the genetic map of an organism to form a transgenic beneficial variety; • Studying the genome sequences and cloning them outside their host; • Studying the configuration and expression of genes. Modern biotechnology creates a lot of challenges in the handling and induction of biotechnological product in an ecosystem. Although the RDT has revolutionized the world with its products in every discipline of life improving the overall standard of human life but on the other hand RDT products have created challenges in the safe handling and administration of RDT products. RDT products can interact with the food chain and

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interact with the overall pattern of ecosystem and community structure. So, biosafety has emerged as a serious challenge in overall scenarios. 1.17 REGULATING RECOMBINANT DNA TECHNOLOGY (RDT)

Despite several uses of RDT the biosafety issues regarding the RDT products are there and the RDT products are the part of debate for their usage or rejection (Gupta, 2000). The RDT may lead to the development of the novel pathogenic varieties which may lead to pandemics and thus diseaserelated issues may become the part of the globe. The biological wars may start, and the globe may lead to self-destruction. The recombinant DNA (rDNA) Advisory Committee of the National Institute of Health, USA (NIH-RAC USA) produced a strict set of rules for working in the RDT labs and issues strict guidelines to work in the biotechnological labs (Nestmann et al., 2002). These rules and terms make the world familiar with the level of containment while working in RDT labs. RDT produced products are being used in various forms across the globe like insulin, human growth hormone, bovine growth hormone, tryptophan, tissue plasminogen activator, and transgenic varieties of plants and animals have been raised. The RDT products pass the strict criteria of reliability, and these products are given authenticity by the Food and Drug Administration (FDA). The USA Department of Agriculture (USDA) along with The Animal and Plant Health Inspection Services (APHIS) issues permits to test a Genetically Engineered product in the field. Any incorrect information provided in the permit application leads to money or imprisonment penalty. 1.18 SOME OF THE PRODUCTS DEVELOPED FROM RDT AND THEIR BIOSAFETY ISSUES 1.18.1 RECOMBINANT INSULIN RDT was first used in the synthesis of human insulin protein from the bacteria on a commercial basis. Food and Drug Authority (FDA) approved the use of insulin to treat the diabetes in 1982. The insulin was prepared from the pancreas of slaughtered animals initially before the advancement of technology (Toke, 2004). The first FDA approved drug is human insulin which was produced by Eli Lilly and Co. under the trade mark of

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“Humulin.” Through RDT the gene for insulin is isolated from the DNA and is inserted into a suitable vector forming rDNA which is introduced in the suitable expression system. The insulin produced by the bacterial expression system is similar to human insulin. The RDT produced insulin replaced the traditional method of purifying the insulin from the animal pancreas and proved as efficient source in the treatment of diabetes. The scientific community however showed its concern in the RDT mediated preparation of insulin and argued that isolated gene may mutate in the process and overall structure of the gene may be altered in the process. Although the amino acid sequence of the Humulin resembles the normal insulin yet, researchers showed their concerns and insulin was subjected for the analysis of risk by Metabolic and Endocrine Drugs in the Center for the Drug Evaluation and Research. 1.18.2 HUMAN GROWTH HORMONE Somatotrophin is the growth hormone that directs the growth pattern in a normal human being. The hormone has been prepared by the biotechnologists to increase the growth rate using RDT synthesis methods. The hormone has been produced by the various pharmaceutical industries. The FDA has a strict set of rules for the administration of the growth hormone in human beings and sets the criteria for its administration due to biosafety issues of the product. The hormone may be misused so there is a restriction in the USA to sell the product on prescription. 1.18.3 BOVINE GROWTH HORMONE The bovine growth hormone, also known as bovine somatotrophin (BST) can also be prepared by RDT. The cows which are provided with the dose of BST produce a greater quantity of the milk. The natural source of the BST is very expensive and time-consuming tasks however the biotechnological advancement has enabled the production of BST in the laboratory using RDT (Toke, 2004). The gene for the BST was isolated and cloned using the expression system of E. coli bacteria. Product is then purified and is transformed into the injectable form. The biosafety issues regarding the safe induction of BST were assessed and it was found that whether

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the cows injected with BST produced by RDT produce the hormone in the milk or not and whether the milk produced by the cows treated with BST is good for human health or not however it was found that the BST is not active in the human and the hormone can be used to increase the milk production in the cows. 1.18.4 RECOMBINANT TRYPTOPHAN

Tryptophan is considered as an essential amino acid and is amongst the 20 amino acids which are the extensively utilized in the protein synthesis. Tryptophan is essential amino acids so it should be supplied to human from the external sources as human body is unable to manufacture it internally. The amino acid tryptophan is used in the biosynthesis of plant hormone auxin and serotonin which is a neurotransmitter. The lack of tryptophan leads to several abnormalities including the unfair mental health and aggression finally leading the person towards suicide. The tryptophan was manufactured by RDT by genetic modification of bacteria, but the people consuming high doses of RDT manufactured tryptophan encountered several health-related issues, and about 37 deaths were reported in America due to using RDT manufactured Tryptophan. It was tested for the biosafety issues, and it was found that about 60 contaminants were the part of the RDT manufactured tryptophan. The biosafety issues regarding RDT mediated synthesis of tryptophan were highlighted and now the tryptophan is synthesized with extreme precautionary measures. 1.19 BIOSAFETY ISSUES REGARDING THE GENE THERAPY Process in which healthy gene is inserted in place of defective gene is called gene therapy. Gene therapy is used to treat several diseases like hypercholesterolemia, cystic fibrosis and coronary artery angioplasty and it holds promise for the treatment of a number of disorders. Despite several advantages the gene therapy possesses some demerits also regarding biosafety and hazardous commercialization. Some of the demerits are being discussed in subsections.

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1.19.1 DELIVERY PROCESS OF GENE AND ASSOCIATED ISSUES Delivering the gene to appropriate cells is a merit of gene therapy but the manipulation of the gene to wrong cells is a demerit of the gene therapy, and several issues regarding the biosafety arise in case of unsafe delivery of a gene towards its target. Unsafe delivery of the gene may lead to the departure of the organism from the normal homeostasis and thus resulting in a malady. 1.19.2 INTEGRATION OF THE DONOR DNA WITH TARGET TISSUES The donor DNA that is being manipulated through the gene therapy should successfully become the part of the recipient cells and should integrate their and express to form its product. If the delivery and integration is inappropriate and unsafe, then disorder may appear, and malfunctioning may result. Sometimes multiple rounds of the gene manipulation are required to address this issue. The genes delivered by the process of the gene therapy require a suitable vector that may be a viral DNA part that may serve as a vector. This viral DNA is treated as a foreign object by the immune system of the target organism, and it may produce certain antibodies in response to it. The immune system may reject the foreign genes as well as the vector being transferred. The vector used to transfer the foreign genes in the genome of an organism should escape from the immune system of the recipient otherwise severe maladies, syndromes, and even death may occur. 1.19.3 DISTURBANCE IN TUMOR SUPPRESSING GENES Sometimes if mishandled the genes delivered may target the tumor suppressing genes of the recipient organism and thus it may induce tumor in the recipient organism. So, the process of the gene therapy should be handled with expertise and proper equipment to avoid any biosafety issue.

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1.19.4 BIOSAFETY ISSUES REGARDING THE INTRODUCTION OF THE TRANSGENIC ANIMALS IN THE NATURAL ECOSYSTEM Animal biotechnology can produce the following concerns and biosafety related issues like: • The impact on the immediate surroundings of the transgenic organism may raise the biosafety issues; • Food items produced by the GMOs are a concern too as they may contain contaminants and allergen with them; • The organ transplantation practices may also cause suppression in the immunity of the recipient organism receiving the xeno-transplant; • The international use of the GMOs should be in containment if a particular GMO has been raised to obtain a pharmaceutical product. Above are some of the concerns which are related to animal biotechnology practices. 1.19.5 SAFETY RISKS ASSOCIATED WITH THE USE OF TRANSGENIC PLANT VARIETIES Following issues may arise if the transgenic species of the plants are introduced in the natural ecosystem: • Plants being used extensively as food and feed may contain some toxins and contaminants in their transgenic forms and they may be harmful in this respect; • All the transgenic varieties being introduced contain some unknown risk they may pose to their surroundings and environment and human are particularly in pearl; • Gene constructs involve the use of the antibiotic resistant markers. The food produced by these plants been produced by the use of antibiotic resistant markers may develop antibiotic resistance; • The transgenic variety if becomes the part of the natural ecosystem flows its genes towards natural biodiversity and thus serious biosafety issues regarding the health of native population may arise;

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•

Above all the society may raise some moral issues regarding the nature of transgenic food (Pushpa, 2006).

Above are some of the major concerns in the field of plant biotechnology and there is need to find the direction in solving the above-mentioned issues. 1.20 GENE FLOW Evolutionary forces like migration genetic drift, selection, mutation, and nonrandom mating are responsible for the gene flow from one population to the other. The change in the gene frequency is a common practice in nature thus evolutionary mechanism operates in the nature (Lesser, 1994). The process of gene flow through the evolutionary forces may transfer the genes from a GM crop to non-GM crop and wild plants (Anuradha, 2005). Such gene flow processes carry environmental risks which include the negative impacts on biodiversity and wild plants may face potential risks on their existence and survival (Bilderbeek, 1992; Asebey et al., 1995). In the environment the gene flow may take three types of modes. The gene may flow from one plant to the other through the pollen grains by the help of insects, wind, and water (Figure 1.2). Secondly the genes may flow through the seeds and various dissemination mechanisms for the seed operate in nature (Lesser, 1994). Thirdly the genes flow may occur by the movement of propagules in the form of vegetative reproductive bodies like tuber and rhizomes. The transgenic plant introduced in the natural ecosystem may transfer the gen to the non-transgenic plants, weeds, and wild relative species (Anderson & Jackson, 2006). The gene flow in the nature between a GM organism and naturally reproducing population may produce health and biological integrity related issues. This may lead to loss of natural biodiversity, which may be replaced by the foreigners very soon and may result in the loss of environmental integrity (Bilderbeek, 1992), compromising the safety of the nature (Figure 1.2). Furthermore, the Trans genes may become the part of the food chains, and intake of their products may raise health related issues in the other organisms, including human beings. Creation of new weeds is also possible due to gene flow.

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FIGURE 1.2 Assessment of all the possible risks. Source: Deepa & Shomini (2013).

1.21 INTELLECTUAL PROPERTY RIGHTS (IPRS) AND BIOSAFETY Intellectual property rights (IPRs) are right of intangible assets of any product produced by any person or company. IPR protects the application of ideas, novel thoughts and information which do have some recognized commercial use. Generally, IPR deals with the patents, trademarks, secrets of trading, ideas, and similar type of rights (Johar & Narnaulia, 2010). The use of biotechnological approaches to make new products from plant, microorganisms, or animal biodiversity in form of useable which do have some commercial values (Senan et al., 2011). The advent and use of biotechnological approaches has led towards the creation of some technical questions of moral and social context which create problems (Pushpa, 2006). IPR is in state of flux in different biotechnological products and these rules are very prominent in US and other technological developed nations, but currently

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other countries of the world do also compete for the biotech markets and importance of IPR has been manifold. Recently World Intellectual Property Organization (WIPO) has been made as permanent chapter of United Nations Organization (UNO) which is clearly addressing the IPR around the globe (Johar & Narnaulia, 2010). This suggests government about the potential IPR problems and their solutions for the biotechnological products. 1.22 INTELLECTUAL PROPERTY PROTECTION (IPP) The world biotechnological products are being protected for: (i) rights of plant races and varieties; and (ii) the patens registered on certain discoveries and inventions. The prerequisite of biotechnological protection in form of “Keeping the secret of Biotechnology” is called protection (Correa, 2005). Protection is very crucial and important in trade and also in biotechnological inventions otherwise it will be mis-used and sell out on a large scale may be useful or harmful for the mankind. The formulation of the law of “copyright” is another form of IPP which is protected by generic code using computer code which is commonly used in industrial products around the world (Correa, 2005; Pollack & Shaffer, 2009). Hitherto, no biotechnologist has claimed the copyright as a form of an IPP. Trademarks are also employed in the protection of biotechnological products and industrial products are being protected and sold with that specific. Trade mark register (TMR) is an advanced form of industrial products which are under IPP, and many companies do follow this paradigm of IPR (Toke, 2004; Correa, 2005). Up to now different plant varieties and cultivars have been given IPR and protected as patents which had been covered under umbrella of International Union for the Protection of New Varieties (UPOV) of plants in year 1961 and 1978. After this UPOV has been effective and many countries of the world follow this mode of IPP. The UPOV protects the 24 plant species as “protectable variety” under rhythm of distinctive, uniform, stable (DUS) and do possess the novelty as well. Later on, patents are form of IPP which is grant of exclusive rights of ownership and selling with trade mark for limited time as new and useful discovery or invention (Chakrabarty, 2002). The grant of patent authority is varying from one country to another, however international procedure and rules are unanimous. The plant patent has been granted under “Plant Patent Act (PPA).” It is known that “transgenic non-human mammals all type whose germ cells and somatic cells comprise of rDNA or chain

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of activated oncogenes sequence pattern introduced into host or desired mammal during the embryonic stage” commonly renown as ‘oncomouse’ (Anderson & Jackson, 2006). 1.23 INTERNATIONAL TREATIES It is known that there three international IPR treatise vague in the world such as: (i) Paris Convention for Protection of Industrial Property; (ii) Budapest Treaty on Industrial Recognition of the Deposit of Microorganism for Patent Procedure; and (iii) Patent Cooperation Treaty (PCT). All laws and rules of property rights protection is governed by these treatises and make framework of IPP (Pollack & Shaffer, 2009). The PCT is commonly recognized as a vehicle for resolving all issues relevant to patents (Chakrabarty, 2002), IPR and IPP through the forum of International Depositary Authorities (IDA) (Correa, 2005). The IPR provides the mechanism of compensating the Fundación for its efforts to address and solve the issues of the biotechnological products (Lesser, 1994). The Fundación requires to be capable to address and disseminate the security and provision of IPR so that the products would not be re-sold. IPR rules are means of influencing which influence the developing countries to keep the diverse sources with a value of rendering the services to world communities (Correa, 1996, 2005). 1.24 REGULATED TRADING IN INTELLECTUAL PROTECTED PRODUCTS (IPP) FROM WILDLIFE IPR and IPP provide regulations for trading the products of wildlife resources which benefit the end users of the indigenous and far farther areas (Pray et al., 2005). It is to regulate the prices of prices in different zone of the country or world, particularly from agriculture commodities (Horbulyk, 1993; Lianchawii, 2005). Currently no mechanism for regulating the trading of these types of commodities is available. However, there are many international agreements present which do provide regulatory mode for these wildlife products in different countries of the world (Lesser, 1994). Internationally two famous treaties life Western Hemisphere Convention (WHC) and the Paris International Convention for Protection of Birds (PICPB) are commonly governing the general rules for the trade of these wildlife and their products around the world. Now these treatises are known as “sleeping treaties” (Pray et al., 2005).

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Now another convention called “Convention on International Trade in Endangered Species of Wild Fauna and Flora” (CITES) is operational in most of the counties. It is practically applied in the conservation of different natural resources of an area. The code of CITES prevents the trade of endangered species or taxa in the world. It has produced a checklist of taxa which are under severe threats, and their trade is prohibited. All countries are made obliged to obey this treaty and imports are governed under this act. The trade is controlled and managed under this rule of CITES and it is called controlling mechanism of trade (Figure 1.3). This CITES treaty not only manages or control freelance movement of plants and animals across borders of different countries but also products or their derivatives are also prohibited under these regulations.

FIGURE 1.3

Bt cotton development process in India.

Source: Deepa & Shomini (2013).

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1.25 CONCLUSION There are a number of issues and concerns regarding the induction of biological hazardous agents in the form of GMOs and biotechnological formulations in the natural ecosystem (Singh et al., 2004). The foreign genes inserted may lead to failure in the expression of endogenous genes with passage of time as silencing the transgenes may result in co suppression of the innate genes. Moreover, the food obtained from the GM organisms and crops may contain contaminants and allergens leading to unknown threats (Anderson & Jackson, 2006). Keeping in view the above concerns, it is important to follow biosafety measures and all of the legislations and protocols must be implemented for the sake of humanity. The anthropogenic influence in the nature may lead to existence of life in pearl. KEYWORDS • • • • • •

biological hazardous agents biosafety clearing house convention on biological diversity genetically modified organisms intellectual property protection intellectual property rights

REFERENCES Anderson, K., & Jackson, L. A., (2006). Transgenic crops, EU precaution, and developing countries. International Journal of Technology and Globalization, 2(1, 2), 65–80. Anuradha, R. V., (2005). Regulatory and Governance Issues Relating to Genetically Modified Crops and Food: An India Case Study. New York, NY: Case Study for the New York University Project on International Governance of Genetically Modified Organism. Asebey, E. J., & Kempenaar, J. D., (1995). Biodiversity prospecting: Fulfilling the mandate of the biodiversity convention. Vanderbilt Journal of Transnational Law, 28(4), 703–754. Bilderbeek, A. S. E., (1992). Biodiversity and International Law. Amsterdam: IOS Press, The Netherlands.

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Chakrabarty, A. M., (2002). Patenting of life forms: From a concept to reality. In: Who Owns Life? Prometheus Books, Amherst, NY. Correa, C. M., (1996). Intellectual property rights and agriculture: Strategies and policies for developing countries. In: Van, W. J., & Jaffé, W., (eds.), Intellectual Property Rights and Agriculture in Developing Countries (pp. 110–113). University of Amsterdam, Amsterdam. Correa, C. M., (2005). How Intellectual Property Rights Can Obstruct Progress. SciDev Net. Deepa, G., & Shomini, P., (2013). IPR, Biosafety and Bioethics (pp. 1–249). ISBN: 9789332514249. Pearson Education Pvt. Ltd., Delhi India. Gupta, A., (2000). Governing Biosafety in India: The Relevance of the Cartagena Protocol. Belfer Center for Science and International Affairs, John F. Kennedy School of Government, Harvard University. Horbulyk, T. M., (1993). Intellectual property rights and technological innovation in agriculture. Technological Forecasting and Social Change, 43, 259–270. Isaac, G., (2002). Agricultural Biotechnology and Transatlantic Trade: Regulatory Barriers to GM Crops. CABI. Johar, A., & Narnaulia, S., (2010). Patenting life in the American, European, and Indian way. Journal of Intellectual Property Rights, 15, 55–65. Lesser, W., (1994). Institutional mechanisms supporting trade in genetic materials. Environment and Trade Series No. 4, UNEP, Geneva. Lianchawii, (2005). Biosafety in India: Rethinking GMO regulation. Economic and Political Weekly, 40(39), 4284–4289. Navneet, A., (2014). A co-dynamic model to frame controversies over genetically modified crops in India. Asian Biotechnology and Development Review, 16(3), 61–85. Navneet, A., (2018). Policymaking in the context of contestations: GM technology debate in India. Studies in Indian Politics, 6(1), 117–131. Nestmann, E., Copeland, T., & Hlywka, J., (2002). The regulatory and science-based safety evaluation of genetically modified food crops: A USA perspective. In: Atherton, K. T., (ed.), Genetically Modified Crops: Assessing Safety (pp. 1–44). London, UK and New York, NY: Taylor & Francis. Pollack, M. A., & Shaffer, G. C., (2009). When Cooperation Fails: The International Law and Politics of Genetically Modified Foods. Oxford, UK and New York, NY: Oxford University Press. Pray, C. E., Bengali, P., & Ramaswami, B., (2005). The cost of biosafety regulations: The Indian experience. Quarterly Journal of International Agriculture, 44(3), 267–289. Pushpa, B., (2006). The social, moral, ethical, legal, and political implications of today’s biological technologies. Biotechnology Journal, 34–36. Scoones, I., (2006). Science, Agriculture, and the Politics of Policy: The Case of Biotechnology in India. New Delhi: Orient Longman Private Limited. Senan, S., Haridas, M. G., & Prajapati, J. B., (2011). Patenting of microorganisms in India: A point to ponder. Current Science, 100(2), 159–162. Singh, B. D., Dey, S., & Singh, B. S., (2004). Conservation of Biodiversity and Natural Resources (pp. 789–812). Daya Publishing House. Toke, D., (2004). The Politics of GM Food: A Comparative Study of the UK, USA and EU. London, UK and New York, NY: Routledge.

CHAPTER 2

Safety and Benefits of Bt and Bt Cotton: Factures, Refute, and Allegations

SURENDRA SINGH SHEKHAWAT, IRSAD, and SAFDAR KAISER HASMI Department of Plant Protection, Aligarh Muslim University, Aligarh, Uttar Pradesh, India

ABSTRACT The advent of the green revolution, which was based on conventional breeding, has undoubtedly increased the crops’ production and nutrition value, and played a crucial role in feeding the current population. However, tracing the population increase and decreasing arable land shows that this is the perfect time to adopt a faster breeding technology based on selecting and incorporating desirable genes. Genetically modified crops (GM crops) produce a superior cultivar at the same time, reducing the time and cost of breeding, increasing the nutritional value and resistance against pest and diseases. Hence, undoubtedly decreasing the cost-benefit ratio and increasing production per unit of area. The GM crops’ potential to mitigate future needs has been successfully proven in Indian cotton and Australian canola. The GM seed market currently has approx. 8.7% CAGR. But at the same time, controversies like the Seralini affair and Monarch butterflies decline are continuously questioning the biosafety of these crops as these crops may lead to the evolution of new potential pest-pathogen weed strains, which may produce more severe threat than the presently available ones. GM crops may produce severe allergens or some severe health issues to human populations. Hence, both the scientific community and

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governmental regulatory bodies should determine these crops’ biosafety by keeping an eye on the environment and human health before releasing them for commercial cultivation. 2.1 INTRODUCTION The green revolution demonstrated a wide array of significance in terms of boosted crop production. However, this was due to achieved by adopting high yielding varieties, agrochemicals, and machinery. Meanwhile, the great extent of heavy inputs unexpectedly resulted in increased pest outbreaks, which elevated dependency on synthetic chemicals. Simultaneously, the broad-spectrum toxicity of these chemical weapons made them more popular among the farmers. Nevertheless, indiscreet application of synthetic molecules resulted in pest resistance, pest resurgence, and lethal effects on beneficial fauna and the environment. Because of the environment and human health concerns, scientists focus on developing sustainable plant protection approaches using biological organisms (Raymond et al., 2011). These organisms (predators, parasitoids, fungi, bacteria, and viruses) work on the principle of natural regulators in the ecosystem and bear a great potential to control both biotic and abiotic stresses. 2.2 HISTORY OF BACILLUS THURINGIENSIS (BT) Shigetane Ishiwatari (1901) first established the association of a grampositive bacteria with “Sotto disease” of silkworm larvae and described this soil-inhabiting bacteria as Bacillus sotto. Later, Ernst Berliner (1911) isolated the same bacteria from the Mediterranean flour moth’s dead larvae at Thuringia state (Germany) and renamed it as Bacillus thuringiensis (Bt). Bt’s insecticidal properties were well known and utilized by the farmers to control flour moths since 1920. Later in 1938, France became the first nation to commercialize Bt-formulation under the trade name Sporein. However, these formulations face several limitations in farmers’ fields, as they could not resist heavy rains, solar UV-rays and toxicity were confined to some Lepidopteran species only. In 1956, Hannay, Fitz-James, and Angus showed the primary cause of Bt’s larvicidal property was parasporal crystal protein. In 1961, Bt was registered as a pesticide by US-EPA (Environmental Protection Agency; Bravo et al., 2011). Another toxic protein, cytolytic protein (Cyt) from the strain – Bt israelensis found

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effective against the dipteran suborder Nematocera and Coleopterans (Osman et al., 2015). In 1970, Dulmage isolated HD-1 strain of Bt from diseased larvae of pink bollworm (PBW), Pectinophora gossypiella. The isolates of Kurstak, studied by de Barjak & Lemille, reported as new subspecies, kurstaki (Sansinenea, 2012). Biopesticides comprise 5% of the total pesticide market, of which 90% are Bt derived (Kumar & Singh, 2015; Seiber et al., 2014) (Table 2.1). TABLE 2.1 Bt Strain Bt aizawai Bt israelensis

Bt kurstaki

List of Popular Bt Subspecies and Their Susceptibility to Insect Orders Susceptible Insects Wax moths (Galleria melonella) Mosquitoes (Aedes aegypti, A. albopictus, Anopheles spp.), black flies (Simulium demnosum) and fungus gnats Caterpillars (Spodoptera litura, Achaea janata, Helicoverpa armigera, H. zea, Thysanoplusia orichalsea, Cabbage looper (Trichoplusia ni), Diamondback moth (Plutella xylostella), Lymantria dispar and Choristoneura fumiferana Larvae of the elm leaf beetle

Bt tenebrionis/ San Diego Bt 4D1 Tomato pinworm (Tuta absoluta) Bt aizawai ABTS Nettle caterpillar (Euprosterna elaeasa) 1857, GC 91, Bt kurstaki HD 1 Bt LSM Fall armyworm (Spodoptera frugiperda)

References Andreeva et al. (2020) Lagadic, & Caquet (2014) Sansinenea (2012); Vimala et al. (2020); Legwaila et al. (2020)

Grantham (2008) Sandeep et al. (2020) Plata-Rueda et al. (2020) Álvarez et al. (2009)

2.3 GENETICALLY MODIFIED ORGANISMS (GMOS) Genetically modified organisms (GMOs) are the genetically engineered organisms (GEOs) with desired traits achieved by inserting specific foreign nucleic acid bacteria, fungi, plants, or other living organisms through Agrobacterium or gene gun (Bawa & Anilakumar, 2013). Recombinant DNA technology (RDT) permits the modification in the genetic makeup of an organism for the desired trait. The application of this technology resolves the challenges faced by the growers while doing conventional breeding. The first GMO was a bacterium, resistant to the antibiotic kanamycin, created by H. Boyer and S. Cohen in 1973. Later in 1974, the world’sworld’s first genetically engineered animal, a mouse,

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was developed by Rudolf Jaenisch by introducing a foreign DNA into its embryo. In 1982, the FDA approved the release of Humulin, insulin produced using bacteria. However, the first transgenic plant (tobacco) was developed in 1982, exhibiting resistance to antibiotic and China became the first country to adopt transgenic plants commercially (Griffitts et al., 2005). Tomato (FlavrSavr) was the first commercially grown GM food crop manufactured and released by Celgene in 1994, known for its’ delayed ripening (Vessey, 2002). Vitamin-A rich Golden rice was the first plant developed to increase nutrient content in 2000 (Chadha et al., 2000). GM crops are also used as bioreactors for the production of medicine and biofuel (Rangel, 2015). 2.4 SIGNIFICANCE OF GM CROPS In 2018, GM crops were grown over the 191.7 million hectares of the land in 26 countries. Total 48.2% of soybean and 13.5% of GM cotton was cultivated in 2019. The 10 Bt crops, cotton, Maise, soybean, eggplant, potato, tomato, rice, sugarcane, cowpea, and poplar, were cultivated on 23.7 m ha area of the world (Anonymous, n.d.). India is the 5th largest global grower of GM crops (6%) after US (39%), Brazil (27%), Argentina (12%), Canada (7%). The adoption rate of Bt cotton in India indicates that technology is benefitting the farming community. The GM crops are highly significant under the following traits: 1. Insect Resistance and Herbicide Tolerance: Crystal proteins (Cry) producing bacteria Bt has excellent insecticidal properties to the Lepidopterans, Coleopteran, and Dipteran pests. It is a species-specific, ecologically viable approach of pest management. The only gene other than Bt is cowpea trypsin inhibitor (CpTI), used in cotton in 1999 (Powell, 2015). Herbicide-tolerant plants are developed by introducing herbicide-tolerant enzyme from a tolerant cell line to a non-tolerant cell line with CMV 35 promotor. A bar gene from Streptomyces hygroscopicers introduced in tobacco, tomato, and potato to produce phosphinotricin acetyltransferase, which inhibits the toxicity of phosphinotricin and bialaphos (Samir & Abbas, 2018). 2. Expression of Coat Protein Genes Against Virus Protection and Antisense RNA: The introduction of coat protein into the plants has resulted in 10–60% inhibition of virus development

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which can be used to develop virus-resistant varieties (Bonny, 2016). Antisense RNA controls the gene expression in organisms by interfering with ribosome binding, transport of mRNA from the nucleus and boosting mRNA degradation. It can be used to prevent the expression of undesirable characteristics (Beachy, 1999). 2.5 NEED FOR GM CROPS 2.5.1 FOOD SECURITY AND SUSTAINABLE PRODUCTION Food security and sustainable agriculture are the prime interest of each country globally. The world’sworld’s population is increasing rapidly, and it will be a challenging task to feed 9.9 billion people by 2050, where agricultural land is continuously diminishing. Therefore, to enhance crop productivity and achieve sustainable production, technological innovation is required (Cuozzo et al., 1988). 2.5.2 PESTICIDAL CONSUMPTION, HEALTH HAZARDS, AND ENVIRONMENTAL EQUILIBRIUM The need for high crop production involves heavy input, which subsequently demands large amounts of pesticides to counter insects and weeds. The manufacture and application of pesticides comprise environmental pollution and the health risk of personnel. Studies conducted on Bt have exhibited wide adoption due to GM crops’ significant increase in crop production in many countries (Anonymous, n.d.). GM crops’ adoption may reduce pesticide poisoning that disrupts the ecological balance, pest resurgence, and negative impact on the non-target organisms and soil microbes (GM Approval Database, 2014; Mishra, 2020; Patton, 2014). Since their introduction in 1996, genetically modified (GM) crops have reduced pesticide use by as much as 581.4 million kg, compared to traditionally grown crop (nearly 8.2% reduction). Bt pesticides have a positive effect worldwide and are being employed against various damage-causing insect pests due to less environmental pollution and the positive impact on beneficial fauna (Betz et al., 2000). Bt cotton is one of the most successful examples of transgenic technology in pest management (Chadha et al., 2000). Because of efficacy, safety, and environmental health, the active protein has been isolated and identified for further mass production to commercialize microbial formulation.

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2.5.3 ALTERATION IN THE NUTRITIONAL COMPOSITION OF FOOD The nutritional status of the crops, fruits, and vegetables can be improved using genetic modification technology (Patton, 2014). Vitamin-rich golden rice and a high amount of methionine in sweet lupine are some of the most suitable examples. It helps in eliminating the malnutritional problem in children of Africa and Asia. Likewise, researchers can modify proteins and carbohydrates’ composition (Dively et al., 2018; Montagu, 1977). 2.5.4 CLIMATE CHANGE, DROUGHT, AND ABIOTIC STRESS These are resulting in significant crop loss of agricultural production. Climate change and temperature rise can alter the insect-pests abundance producing a negative impact on the farmer’s economy. Scientists have made possible efforts to solve drought-resistant varieties for dryland areas to increase sustainable crop production and degraded land could be transformed into fertile land by incorporating mycorrhiza (Lu et al., 2012). 2.5.5 DEVELOPMENT OF GM CROPS After the discovery of the DNA model, understanding of genetic engineering is significantly increased. RDT is a famous laboratory tool to manipulate the genetic structure. It involves the following steps (Rizzi et al., 2012): • DNA extraction from the organism for the desired trait; • Gene cloning: isolation and mass production of the target gene in a host cell; • Gene packaging and amplification; • Transformation: using a gene gun or through Agrobacterium; • Testing for the gene expression/back cross; • Assessment of safety measures and regulatory approval. 2.5.6 DEVELOPED GM CROPS Glyphosate-tolerant soybean, first released in 1996, now acquired almost 90% of the total cultivated area in the U.S. Oleic acid soybean contains 80% monounsaturated oleic acid than conventional varieties (24%). GM corn inherits traits to resist herbicides and insect pests, reducing weed and insecticide spray pressure to the crop. GM canola is also herbicide-tolerant

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and contains low levels of saturated fats and a high level of laurate, possessing qualities of palm oil and coconuts as well as high in oleic content (Table 2.2). TABLE 2.2

List of Events in Developing GM Crops

GM Crop Soybean Corn Canola

Significant Trait Herbicide tolerance, oleic acid-rich Herbicide tolerance, insect resistant Herbicide tolerance, rich in oleic acid and laurate, resistance to white mold Cotton Herbicide tolerance, insect resistant Potato Insect and virus-resistant Squash Resistant to watermelon mosaic virus and Zucchini mosaic virus Tomato Extended shelf life Papaya Resistant to papaya ringspot virus Alfalfa Herbicide tolerance, higher RFQ and NDFD Arctic apple Prevents browning after the cut Bean (Phaseolus vulgaris) Resistant to golden mosaic virus Carnation Modified flower color Chicory Large scaled production of artemisinin (a drug for malaria) Cowpea Resistant to pod borer (Maruca vitrata) Aubergine Resistant to fruit and shoot borer Eucalyptus Fast growth and increased wood production Flax Tolerant to herbicides Melon Delayed senescence Petunia Modified flower color Pineapple Pink pineapple Plum Resistant to plum pox virus Poplar Easy industrial processing Rice Resistant to herbicides, insect–pests and flood, golden rice (Vitamin A rich) Rose Modified blue color petals Safflower The high oleic acid content Sugar beet Herbicide tolerance Sugar cane Increased productivity and ethanol production Sweet pepper Resistance against CMV Tobacco Ethanol production Wheat Herbicidal resistance, drought tolerance Source: Compiled from: Schmidt et al. (2008); Pagano (2014).

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2.6 APPLICATION OF BT IN GM CROPS Field trials of Bt cotton were started in India by MAHYCO (Maharashtra hybrid seed company) under the Department of Biotechnology’s supervision and the Ministry of environment and forest. The DBT constituted an expert committee genetic engineering approval committee (GEAC) to approve GM products in the country. In the Indian context, research and development in GMOs are described or elaborated in a flow chart by implementing a regulatory mechanism. Scientists have been carrying out several studies on developing broad-spectrum Bt crops to support life and vision to improve plant health using GM. Hence, GMOs are efficient in crop production and protection effectively and environmentally safe throughout the world. Most of the Bt approved crops are cotton and corn (Jayaraman, 2002) (Table 2.3). TABLE 2.3 The List of Bt Approved Plants Protein

Target Pest

Cry1Ab

Lepidoptera

Cry1Ac

Lepidoptera

Approved Registered Products Crop Maise YieldGard (Monsanto), Agrisura CB/LL (Syngenta) Bollgard (Monsanto) Cotton Maize

Bt Xtra (Monsanto)

Soybean

Intacta Roundup Ready 2 Pro (Monsanto)

Brinjal

Cry1Ac + Cry1F

Lepidoptera

Cotton

Cry1Ac + Cry2Ab2 Cry1A.105 + Cry2Ab2 Cry1Ab + Cry2Ae Cry1Fa2 Cry3Bb1

Lepidoptera Lepidoptera Lepidoptera Lepidoptera Coleoptera

Soy Cotton Maise Cotton Maise Maise

mCry3A Coleoptera Cry34Ab1+ Cry35Ab1 Coleoptera eCry3.1Abd Many

Maise Maise Maise

BARI Bt Begun-1, -2, -3, -4 (MAHYCO) White streak (Dow) DAS-81419-2 (Dow) Bollgard II (Monsanto) Genuity VT Double Pro (Monsanto) TwinLink (Bayer) Herculex I (Dow) YieldGard Rootworm RW (Monsanto) Agrisure RW (Syngenta) Herculex RW (Dow and Dupont) Agrisure Duracade (Syngenta)

Source: Obtained data from: http://www.isaaa.org/gmapprovaldatabase.

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A specific type of modification has been considered when the whole Bt toxin Protein domain is integrated with another Cry protein domain (Hofte & Whiteley, 1989; Nakamura et al., 1990; Honée et al., 1991). Domain exchange has exhibited a positive response in changing native Cry protein activity against a new devastating target pest. For instance, the swapping was performed to modify the spectrum activity of Cry protein, Cry1A.105 and Cry1Ab/Ac; demonstrates superior performance toward fall armyworm larvae (Spodoptera frugiperda) but otherwise retained specific activity toward other target lepidopteran pests (Donovan et al., 1992) (Table 2.4). TABLE 2.4

Specificity of Crystal Proteins Against Insect Group

Type of Crystal Protein Cry1A, Cry1Ab, Cry1Ab-Ac, Cry1Ac Cry1Ca, Cry1Da, Cry1Fa

Insect Order/Species Lepidoptera; Spodoptera frugiperda Lepidoptera; Spodoptera exigua Cry1Fa2 Fall armyworm mCry1F Lepidopterans in Maize Cry2 Diptera and Lepidoptera Cry2Ab2 + Cry1F + Cry1A.105 Lepidoptera and Coleoptera (pyramided in corn) Cry2Ae Helicoverpa Cry3Aa Coleopteran; Rhyzopertha, Cylas, Chrysomelidae, and Tenebreonidae families Cry3Ba Diabrotica undecimpunctata Cry3Ca

Leptinotarsa decemlineata Cylas brunneus

Cylas puncticollis mCry3A Coleoptera; western corn rootworm Cry4 Diptera Cry5 Coleopteran and Lepidoptera Cry3A, Cry34Ab1, Cry35Ab1, Coleoptera; corn rootworm Cry3Bb1 mCry51Aa2.834_16 Hemiptera; Lygus Hesperus and L. lineolaris Cry9C Ostrinia nubilalis, Diatraea grandiosella Cry51Aa2.834_16 Frankliniella fusca

References Ríos-Díez et al. (2012) Hernández-Martínez, Ferré, & Escriche (2008) Ojha et al. (2014) Illrich et al. (2008) – Marques et al. (2019) – Ekobu et al. (2010); Oppert, Morgan, & Kramer, (2011); Park et al. (2009); Lopez-Pazos et al. (2010) Donovan et al. (1992) Ekobu et al. (2010) Walters et al. (2008) Ben-Dov (2014) WHO (n.d.) – Hester & Hester (2018) Reed & Halliday (2001) Dhillon & Sharma (2013)

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2.7 ACTION MECHANISM OF BT Bt acts as a gut poison, so it must be ingested along with food. After reaching into the gut, crystal protein of 130–140 kDa size broken down to 60–70 kDa by proteolytic enzymes at 8–10 pH. Broken particles penetrate the peritrophic membrane and bind with specific receptors present on the microvilli. Due to pores, toxin penetrates the cell and swollen it, followed by separation of the basement membrane and midgut epithelium. Further, gut juices of high pH spread into the hemocoel of insect and cause septicemia. Insects devoid of receptors in gut epithelial cells remain unaffected by Bt (Melo et al., 2016).

2.8 BT COTTON Cotton (white gold) is an important cash crop and primarily used as raw material for the textile industry. India is the leading producer of cotton followed by China, the USA and Pakistan (Khan et al., 2020). In India, cotton production was recorded 32.80 million bales (1 bale = 170 kg) in 2017–2018. Insect–pests are the major limiting factor in reducing the yield and quality of the cotton crop. Cotton crop is being attacked by 16 major

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insect species of sucking and borer pest complex (Rajendran, Birah, & Burange, 2018). Bollworm complex can minimize the yield loss of 67%. Bt cotton is a genetically engineered crop with Bt bacteria capable of producing toxin proteins (Cry) to mitigate lepidopteran pests. It was first approved for cultivation in 1995 in the USA and 2002 in India. The area under Bt cotton was 93.5% of total cotton cultivated land with 387 kg/ ha productivity. Area and Bt cotton production in India increased by 1.46 and >2 fold, respectively (DAC & F.W.). Gujrat, Maharashtra, Telangana, Rajasthan, Madhya Pradesh, and Haryana are the leading cotton producer states (pib.gov.in). The bollworm complex produces a 30–40% loss in India (Faisal et al., 2012) (Table 2.5). TABLE 2.5

List of Significant Cotton Insect–Pests

Sucking Pest Complex Leafhopper (Amarasca devastans) Whitefly (Bamisia tabaci) Thrips (Thrips tabaci) Aphids (Aphis gossypii) Mealybug (Phenacoccus solenopsis)

Borer Complex Tobacco caterpillar (Spodoptera litura) Pink bollworm (Pectinophora gossypiella) Spotted bollworm (Earias vittella) Cotton bollworm (Helicoverpa armigera) –

Source: NCIPM; Mohan et al. (2014).

2.9 ALLEGATIONS ON PRODUCTIVITY AND PROFITABILITY OF BT COTTON Many controversies have arisen since the adoption of Bt cotton in India regarding its efficacy, productivity, and sustainability. Stone (2012) alleged that some researchers and authors created the narrative of the technological triumph of Bt cotton in India (Davis, 2012) was replied by Herring (2014). Kranthi & Stone (2020) concluded that the introduction of Bt cotton in India didn’t make a positive impact (Kranthi & Davis, 2020) in reply to Qaim’s (2020) Bt cotton, yields, and farmers’ benefits (Qaim, 2020). These controversies are stuck in a loop of questions and answers and still not reached to any conclusion. Here is a review on the cultivation of Bt cotton claiming a positive impact on the profitability. Bt cotton’s introduction resulted in minimized resource input, with decreased pesticide spry and health hazard to farmers and beneficial species. It uplifted the livelihood of more than 6 million small stakeholder farmers of India by providing high production (ISAAA,

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2018). A study by Kathage & Qaim (2012) found that Bt cotton has increased up to 50% profit, 24% yield increment, and 18% upward trend in households’ consumption expenditures (Kathage & Qaim, 2012). Comparing total input cost and net profit of 4 states in India (Andhra Pradesh, Gujrat, Maharashtra, Tamil Nadu) between Bt and non-Bt cotton revealed Bt is more profitable in all states except A.P. The CB (cost: benefit) ratio of Bt was recorded high in GJ (2.150 Bt, 1.811 non-Bt) and percent net profit from Bt over non-Bt was high in TN (175.46%) followed by M.H. and GJ (67.40 and 56.07%) but, found negative in A.P. (0.75%). Bt cotton yields 30.71% more and lowers pesticide application by 23.98%. Around 57.6% of sampled farmers responded that the seed rate of Bt was reduced by 25%, 96% said that Bt was resistant to bollworm attack, 90% were willing to grow Bt cotton. In Gujrat, 88.89% of farmers seem Bt have an advantage in reducing pesticide cost and improving the economy; 54.44% claimed a substantial advantage, while 38.89% agreed on Bt’s benefit in yield. In Maharashtra, 70% of farmers have reported no infestation of the bollworm complex. The 66.23% of farmers expressed that the Bt cotton has a greater number of bolls, 71.56% claimed better yield and 87.8% experienced bigger boll size than non-Bt crop (Gandhi & Namboodiri, 2009).

2.10 BENEFITS: IN REDUCING POLLUTION, PESTICIDE APPLICATION, AND HEALTH HAZARDS Cotton accounts for 5% of the total cropped area but utilizing half of the total pesticides solely (Bhardwaj & Sharma, 2013). GM crops are undoubtedly helping the farming community by uplifting their economic and

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social status, reducing pesticide hazard, and securing world food safety by providing a highly productive. A long nine-year study of GM crops suggested that the technology effectively reduces 172 million kg of pesticides (14%) and decreased greenhouse gas emissions equal to removing 5 million cars from the road (Brookes & Barfoot, 2005). Impact of Bt cotton on the farmer’s health in Pakistan reportedly saved 7 million U.S. dollars annually (Kouser, Spielman, & Qaim, 2019). Subramanian & Qaim (2009) analyzed the effect of Bt technology on the rural economy. They found 34% more yield, 50% reduction in overall insecticide consumption, including a 63% decrease of bollworm complex in Bt over conventional varieties (Subramanian & Qaim, 2009). A long-term analysis by Krishna & Qaim (2012) on the effect of Bt cotton adaptation on multiple factors (yield and pesticide reduction) found significantly positive. They found an overall decrease of 30 million kg pesticides each year comprising 37% reduction and 35% yield increase in 2002–2004 and 50% in 2006–2008 (Krishna & Qaim, 2012). Introduction of Bt cotton benefitted environment resulted in higher ecosystem impact quotient (EIQ) increased to 68% in 2006–2008 was 39% in 2002–2004 (Veettil, Krishna, & Qaim, 2014). The introduction of Bt cotton in Australia showed a significant 97% reduction in the insecticidal application. Before introducing Bt cotton, the crop required to spray 10 to 14 times reduced to 0–3 sprays only. Bt cotton and reduction in pesticides usage collectively resulted in the implementation of IPM practices, better soil health, increased quantity of beneficial fauna, improved yield, minimized pollution and health risk. An average income gain of 180 dollars/ha, with 395 million dollars overall profit, was reported (Wilson et al., 2013; Cotton Australia, n.d.). Decrease in the average number of sprays in non-Bt (8.11) to Bt (4.27) reported by Gandhi & Namboodiri (2009). In Argentina, it has been reported to reduce insecticides application to 50% (Qaim & De Janvry, 2005). In China, Bt cotton also helped the 4 million small farmers produce more income by minimizing pesticides input (Pray et al., 2002). Reduction in overall usage of pesticides in cotton was reported to 22.9% from 1996 to 2006 (Naranjo, 2011). 2.11 ALLEGATIONS ON THE RESURGENCE OF SECONDARY PESTS Bt was primarily developed to inhibit the Lepidopteran borer complex in the cotton, which reduced broad-spectrum synthetic insecticides’ dependencies. The decreased insecticides spray and interspecific competition

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between species resulted in some minor pests’ resurgence reported in studies, but they can be managed. Dhillon & Sharma (2012) studied Bt cotton’s effect on arthropod abundance and yield of cotton. The study found that the population of American bollworm (H. armigera) on Bt cotton was found lower (4 larvae/100 plants) in comparison to non-transgenic (10.4 larvae/100 plants) variety along with high mature boll openings (9.6/plant). Reduction in boll damage in Bt (12.8%) compared to non-Bt (40.2%). The cotton jassid population (Amarasca biguttula biguttula) was reported lower, but the whitefly (Bemisia tabaci) population was high on Bt (65.2/100 plants) over non-Bt (45.6/plants). The influence of natural enemies and parasitization was found similar in both crops. Although, Cry1Ac protein was tressed in Cheilomenes sexmaculatus, chrysopids, cotton jassids, Thrips tabaci, Myllocerus spp., Oxucarenus laetus, spiders, bugs, and grasshoppers. Reduced insecticide application helped in the resurgence of secondary pests; Spodoptera litura, mealy bugs, thrips, aphids, leafhoppers, and leafminors but impacted positively on bee population in Punjab, Haryana, and Rajasthan (Dhillon, Gujar, & Kalia, 2011). Lawo et al. (2009) found in a study that feeding by aphid on Bt cotton didn’t show any significant effect on the performance except a difference in the sugar composition of honeydew (Lawo, Wäckers, & Romeis, 2009). The application of systematic pyrethroids reduced the beneficial fauna population in a cotton agroecosystem (House et al., 1985). A study regarding the occurrence of arthropods between Bt (47 families) and non-Bt (55 families) cotton represented minor or non-significant difference (Sisterson et al., 2004). Hagenbhucher et al. (2013) associated the cotton aphid population in Bt crop and secondary metabolites. He stated that the reduced level of terpenoids in Bt cotton benefits the population increase of aphids in cotton (Hagenbucher et al., 2013). A study correlated between cotton aphid and Bt resulted that the Bt + CpTI cotton produced shorter and prolonged life, reduced survival with high fecundity in a first and second generation while in Bt, the more extended reproductive period in the first generation and higher survival in 3rd generation (Liu et al., 2005). Introduction of Bt successfully controlled the bollworm complex, but secondary pests become major (Wang et al., 2008). The secondary pest outbreak has been reported from South-eastern USA, where Nezara viridula and Euschistus servus’s population found high (Zeilinger, Olson, & Andow, 2016).

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A study in China found the secondary pest of cotton, spider mites (Tetranychus cinnabarinus), become a severe pest of cotton fields (Ma et al., 2014). The study also supported by Shudong et al. (2003) reported increased spider mite population in Bt cotton than non-Bt crop, but fewer aphids were recorded. He also found the high activity of natural enemies in Bt cotton over non-Bt (Shudong et al., 2003). Before introducing Bt in China, the minor pest-mirid buds get controlled by broad-spectrum insecticides utilized for cotton bollworm. But, since Bt’s introduction avoids the spray, it resulted in a new major pest problem (Betzet al., 2000; Baptiste, Agnès, & Ricroch, 2010). Bt cotton impacted the two predator species, Orius tristicolor and Geocoris punctipes, negatively by decreasing adult survivorship by 27–28% while no effect on Nabis sp. and Zelus renardii (Ponsard, Gutierrez, & Mills, 2002). The secondary pest outbreak can be solved by adopting biological control and integrated pest management (Naranjo & Ellsworth, 2010). Around 15 years of the study concluded that Bt cotton successfully benefitted the farmers to counter the lepidopteran pests with decreased pesticidal consumption. No outbreak of secondary was reported in China (Qiao, Huang, & Wang, 2017). Zhao et al. (2011) found that insecticidal consumption possibilities regarding secondary pest issues were negative and lower than earlier studies (Zhao, Ho, & Azadi, 2011). The same reason was also found for the repeated outbreaks of Apolygus lucorum in North China due to avoidance of insecticidal sprays in Bt cotton (Li et al., 2011). 2.12 ALLEGATIONS ON RESISTANCE DEVELOPMENT AND MANAGEMENT Development of resistance against Bt has been reported in several laboratory studies, but field resistance observed in India only. Resistance development is common phenomena in insects, and the same happens with pesticides also. The rate of resistance development in insects is proportional to higher fecundity and voltinism. So many factors are involved in resistance development, and it is not unique to Bt. In India, high temperature and climatic conditions favor an increased population of insects. Several practices are suggested to deal with resistance are use of refugees – providing natural host or cultivation of non-Bt crop on 20–25% area, gene pyramiding – the combination of two or more genes to counter the target pest implement of IPM or biological control.

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The development of field resistance against Bt reduces its efficacy, and till now, 19 cases have been reported globally (Tabashnik, Carrière, & Gassmann, 2019). Brevault et al. (2015) reported seed mixtures refuges producing 2–4.5-fold more resistance than separate blocks for Helicoverpa zea (Brévault, Tabashnik, & Carrière, 2015). A study conducted during 2002–2003 in Arkansas state conformed that larvae surviving on Bt have the resistance capacity compared to data recorded in 1992–1993 before Bt’s release (Luttrell et al., 2004). The resistance of PBW to the Bt cotton was reported from China’s Yangtze River valley but not for the total failure of control. A decreased trend has been observed in Bt’s susceptibility to PBW from 2005 to 2010, LC50 become greater, survival rate was increased 0 to 8.6% in the year 2008, 2009, and 2010 (Wan et al., 2012). The bollworm remained susceptible to Bt in America and China but found strong resistance in Western India. Larva susceptible to Bt could not complete the development, but 66.1% larval recovery shows high resistance in Gujrat and adjoining cotton-growing areas. Laboratory analysis resulted in more than 2,000-fold less susceptibility (Mohan et al., 2014; Ojha et al., 2014). PBW is a major devastating pest of cotton found to be broadly diversified genetically (Naik et al., 2020), which can also survive pyramided Bt cotton with Cry1Ac, Cry2Ab protein (Fabrick et al., 2015). The first report of resistance was observed from Gujrat in 2008 against Cry1Ac (Dhurua & Gujar, 2011). Nair et al. (2016), in laboratory analysis of Bt diet with PBW, found a 257-fold or 94% increase in LC50. Resistance against PBW in North India is not reported in central and Southern parts; larval recovery was found high at 72.49% from Bt II. LC50 for Cry1Ac found increased from 0.33 µg/ml in 2013 to 6.938 µg/ml in 2017 and for Cry2Ab was 0.014 µg/ml in 2013 to 12.51 µg/ml in 2017 (Nair et al., 2016). 2.13 RESISTANCE MECHANISM Fabrick et al. (2009) reported three alleles linked with resistance development against Bt were (r1, r2 and r3) on cadherin (Fabrick, Jech, & Henneberry, 2009). The high degree of PBW survivorship in Bollgard was found due to reduced binding of crystal proteins to receptors on the brush border membrane (Ojha et al., 2014). Wang et al. (2020) showed a genetic connection between the resistance of PBW to Bt, reported the r14 allele of PgCad1 (bollworm cadherin gene) lacking 36 amino acids in CR5 (cadherin

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repeat 5). This PgCad1 protein is found majorly on the brush border of susceptible larvae and r14 creates resistance by lowering the translation with increased degradation (Wang et al., 2020). Another study by Fabrick et al. (2014) suggests that mRNA splicing involved in the field-evolved resistance against Bt (Fabrick et al., 2014). Resistant larvae showed less abundance of PgCad1 protein and 79–190 times lesser transcription of this gene (Fabrick et al., 2020). The ATP binding cassette gene-PgABCA2 was found responsible for the loss of exon six due to alternative splicing that produces resistance against Cry2Ab protein in India and U.S. populations (Fabrick et al., 2020). Resistance is also associated with the cadherin transmembrane mutation, which interferes with cadherin’s transportation to the cell membrane (Wang et al., 2018). Association between long noncoding RNA (lncRNA) at intron 20 of cadherin alleles influencing Cry1Ac. Small interfering RNAs (siRNAs) can minimize transcription of both lncRNA and PgCad1, resulting in lower susceptibility to Cry1Ac (Li et al., 2019). First Bt cotton (Bollgaurd I) was released in 1996 by Monsanto. An indigenous insect-resistant cotton variety, Bt-Bikaneri Narma (BNBt), was developed by NRCPB, New Delhi, UAS Dharwad, and CICR Nagpur from Gossypium hirsutum L incorporating the Cry1Ac gene (Mayee et al., n.d.). Lowrie et al. (2020) reported multiple factors involved in the resistance mechanism against Bt cotton, i.e., increased expression of trypsins and serine proteases, low expression of aminopeptidases and cadherins and three immune pathways (jak/STAT, Toll, and IMD) also demonstrated a significant variation in expression (Lawrie et al., 2020). 2.14 RESISTANCE MANAGEMENT To overcome this problem, specific tactics were developed to delay the resistance. These are refuge concept – growing of non-Bt crop (on 25% area) with Bt and release of the sterile male population (Tabashnik et al., 2012). Around 21 years of the long-term study showed that the PBW could be successfully countered by adopting a male-sterile mass release in Bt cotton. It reduced the estimated population of 2 billion in 2005 to zero in 2013 in Arizona state and saved farmers’ 192 million dollars with an 82% reduction in consumption of insecticides (Tabashnik et al., 2020). The hybridization of Bt with non-Bt cotton and using the seeds of F2 generation resulted in the minimizing resistance against PBW (Wan et al.,

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2017). To counter Bt-resistant H. armigera, next-generation cotton incorporating RNAi has been developed, which interferes with JH (juvenile hormone) synthesis altering JH acid methyltransferase and JH binding protein (Ni et al., 2017). Pyramiding: containing two or more Bt toxins against the same pest, including Bt toxin and RNAi from dsRNA of H. armigera, predict a delay in resistance by 14 to 75 years (Ma & Zhang, 2019). 2.15 ALLEGATION ON THE ROLE OF BT COTTON IN FARMERS’ SUICIDES IN INDIA Suicides of farmers are allegedly linked to the adoption of Bt cotton and biotechnology. Gruere & Sengupta (2010) denied it and found no change in the trend of farmers’ suicide and Bt cotton introduction (Gruére & Sengupta, 2011). An analysis in DNA by Manjunath (2012) reported the reason behind the suicides were – crop failure, lack of water and financial crisis (Manjunath, 2012). 2.16 ALLEGATIONS ON THE NEGATIVE IMPACT OF BT ON SOIL MICROBES AND BIODIVERSITY Residues of Cry1Ac protein in the soil were not found by growing Bt cotton in the fields (Head et al., 2002). Bt plant is equivalent to its counterpart in all qualities and contents except one gene expression-Bt. Rather than harming the organisms, it encourages the beneficial fauna, which is supposed to be suppressed by intensive use of pesticides. EPA conducted several experiments for the safety investigation of existing registered microbial pesticides and reported no deposition of Cry protein occurs in soil. Soil salinity can affect the expression of Cry protein in the cotton leaves. Bt protein’s efficacy reported decreasing with increased soil salinity (Luo et al., 2017). 2.17 TOXICOLOGICAL SAFETY ASSESSMENT OF BT MICROBIAL FORMULATIONS IN AGRICULTURE There is a comprehensive history of safety concern for Cry protein for human consumption (Hammond et al., 2013). Cry proteins are selective

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to gut receptors, were not found in non-target insects, humans, and other vertebrates. Bt microbial formulations have been continuously employed against mosquitoes’ immature larvae in stagnant water (Organization, 1999; Bravo, Gill, & Toxicon, 2007) and to overcome the insect pest of grown vegetables. Bt classified as biopesticides and claimed safe to use by the US EPA (2015). There is an essential characteristic of narrow biological activity and considered intrinsic least toxic compared to traditional pesticides (EPA, 2015). Therefore, Potential menace related to GM crops needs to be determined for the safety purpose of human health and associated flora and fauna. GM/Bt products’ consumption is safe for humans with no noticeable adverse effects (Federici & Siegel, 2008). 2.18 SAFETY ASSESSMENT OF BT ON HUMAN HEALTH Several regulatory agencies have reported that GM crops are safe for human consumption. The United States EPA (1998b) advocated the use patterns for Bt may result in dietary exposure with probable residues of Bt spores on raw agricultural products. However, without any toxicological worries, the risk is not expected in both the general adult population and children when Bacillus treated products consumed by the distinct population segments. Likewise, WHO/IPCS also declared that Bt has not reported causing any antagonistic or contrary effect on human health while drinking Bt containing water and foods. Further, human health was confirmed through Bt ingestion test; volunteers were exposed to the heavy dose of Bt spore (1010) for five days to detect the negative effect on the victim’s health or inhalation of Bt spores at an amount of 100 mg/day for five days (Siegel, 2001). Therefore, for many decades, Bt formulations have been excluded from the necessity for a maximum residue level in countries where they have been listed (OECD, 2007) that there is no withdrawal time required for consumption of Bt microbial sprayed crops. The world’s top health agencies (WHO/FAO, OECD) have been recognized as the safe application of Bt crops (Cry protein) in Agriculture. Hammond et al. (2013) illustrated some critical features in the context of chronic toxic effects on human health (Gardner, 1988): i. Digestive GI tract enzymes play a vital role in breaking down the protein macromolecules into small peptides, facilitating absorption. Moreover, there is an inverse relationship between molecular

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size and absorption (smaller molecules absorb more rapidly than larger ones) (Gardner, 1988). As a result, the capacity for absorption of an intact protein from the GI tract is much lower than low molecular weight. ii. Multiple barriers are actively involved in restricting the passage of intact proteins into cells after dietary consumption (Kier & Petrick, 2008). The specific membrane contains receptors and transporters that assist the unchanged uptake of a particular protein. iii. GM food ingestion didn’t show any carcinogenic or mutagenic problem (Pariza & Foster, 1983; EPA, 2000; Pariza & Johnson, 2001).

2.19 ECOLOGICAL RISK ASSESSMENT Presently, most GM crops are grown with desirable properties of plants such as Insect pest resistance and herbicide resistance. Cry protein significantly reduces pest activity. If farmers apply other strategies to overcome weeds and insect pest management, they need to put more amount and labor-intensive techniques (Bawa & Anilakumar, 2013) (Table 2.6). 2.20 CONCLUSION Undoubtedly, the need for superior cultivars is growing daily to cope with the upcoming hunger threat due to the increasing population load on shrinking cultivable land. The rising environmental concern due to heavy pesticidal load on cultivated crops also strongly recommends adaptation and popularization of self-immune crops that can combat the biotic and abiotic stresses with little or no impact on environmental health. Up looking at all these facts and needs, GM crops are the future of agriculture due to their relatively faster development and low-cost improvement mechanisms. Many scientific and government data are available that strictly support GM crops and products’ success and safety. The most notable successes story of genetically engineered crops belongs to Bt-cotton (India) and Bt-brinjal (Bangladesh). Bt-cotton’s introduction is one of the most dominant reasons behind the boosted production of cotton balls in India, which lend a hand to the Indian cotton industry to become an exporter rather than an importer.

Commercially Available Bt Products

Manufacturer (Company) Valent Bioscience Corp.

Product

Bt Strain

Applicable Target Pest Unit Dipel WP 16,000 BIU/mg Gypsy moth (Lymantria dispar), cabbage looper B. thuringiensis (Trichoplusia ni), Spruce budworm, bagworm (Choristoneura fumiferana) Thuricide48 LV B. thuringiensis 12.7 BIU/L Lymantria dispar, Choristoneura fumiferana Vectobac200 G – B. thuringiensis israelensis – Novodor flowable B. thuringiensis spp. 3.6% Leptinotarsa decemlineata Concentrate tenebrionis AFA Environment Inc. Aquabac II, XT B. thuringiensis israelensis 1,200 ITU/mg Mosquitoes (Aedes aegypti) and black flies AFA Environment Inc. Dipel 176 – – Forest tent caterpillar (Malacasoma disstria), Lymantria dispar, and Choristoneura fumiferana AEF Global Inc. Bioprotec aqueous – 12.7 BIL/L Lymantria dispar, Choristoneura fumiferana biological

Safety and Benefits of Bt and Bt Cotton

TABLE 2.6

Note: ITU: International toxic unit; IU: International unit. Source: Modified from: Brar et al., (2006).

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Similarly, the Bt-brinjal in Bangladesh boosted brinjal production after the legal introduction of four Bt-varieties in the farmers’ field. However, there are few negative impacts also been observed by researchers, which cannot be ignored, but still, the positive side wins the debate. As the negative traits associated are merely unrelated as the technology was quite successful in obligating the threat they were implemented, the Bt-cotton was explicitly developed to control bollworms. However, according to the allegations, it seems that it will kill everything else except the bollworms. The battle between GM crops supporters and opponents seems endless; however, their popularity and adoption are relatively unaffected. Simultaneously, all the available scientific data refute the opponents’ various allegations and demonstrate the right technology to farmers and the country. However, it is mandatory to strictly check all the aspects of human and environmental hazards before releasing any new technologies commercially to farmers, and from time to time, re-assessment should also be made to contain any undesirable change. KEYWORDS • • • • • •

Bacillus thuringiensis Bt-cotton cowpea trypsin inhibitor cry genes ecosystem impact quotient genetically modified crops

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Anonymous. Bt Insect Resistant Technology. ISAAA https://www.isaaa.org/resources/ publications/pocketk/6/default.asp (accessed on 14 October 2022). Anonymous. New Plant Variety Consultations. https://www.cfsanappsexternal.fda.gov/ scripts/fdcc/index.cfm?set=NewPlantVarietyConsultations&sort=Date_FDA_Letter_ Completion&order=DESC&startrow=151&type=basic&search= (accessed on 14 October 2022). Baptiste, J., Agnès, B., & Ricroch, E., (2010). Emergence of minor pests becoming major pests in GE cotton in China: What are the reasons? What are the alternatives practices to this change of status? GM Crops, 1, 214–219. Bawa, A. S., & Anilakumar, K. R., (2013). Genetically modified foods: Safety, risks and public concerns: A review. J. Food Sci. Technol., 50, 1035–1046. Beachy, R. N., (1999). Coat-protein-mediated resistance to tobacco mosaic virus: Discovery mechanisms and exploitation. Philos. Trans. R. Soc. B Biol. Sci., 354, 659–664. Ben-Dov, E., (2014). Bacillus thuringiensis Subsp. israelensis and its dipteran-specific toxins. Toxins, 1222–1243. Betz, F. S., Hammond, B. G., & Fuchs, R. L., (2000). Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests. Regul. Toxicol. Pharmacol., 32, 156–173. Bhardwaj, T., & Sharma, J. P., (2013). Impact of pesticides application in agricultural industry: An Indian scenario. International Journal of Agriculture and Food Science Technology, 4, 817–822. Bonny, S., (2016). Genetically modified herbicide-tolerant crops, weeds, and herbicides: Overview and impact. Environ. Manage, 57, 31–48. Brar, S. K., Verma, M., Tyagi, R. D., & Valéro, J. R., (2006). Recent advances in downstream processing and formulations of Bacillus thuringiensis based biopesticides. Process Biochem., 41, 323–342. Bravo, A., Gill, S. S., & Toxicon, M. S., (2007). Mode of Action of Bacillus Thuringiensis Cry and Cyt Toxins and Their Potential for Insect Control. Elsevier. Bravo, A., Likitvivatanavong, S., Gill, S. S., & Soberón, M., (2011). Bacillus thuringiensis: A story of a successful bioinsecticide. Insect Biochemistry and Molecular Biology, 41, 423–431. Brévault, T., Tabashnik, B. E., & Carrière, Y., (2015). A seed mixture increases the dominance of resistance to Bt cotton in Helicoverpa zea. Sci. Rep., 5. Brookes, G., & Barfoot, P., (2005). GM Crops: The Global Economic and Environmental Impact—The First Nine Years 1996–2004. Farm Income Effects. Chadha, K. L., Ravindran, P. N., Sahijram, L., & Swaminathan, M. S., (2000). Biotechnology in Horticultural and Plantation Crops (Vol. 1, pp. 1–25). Malhotra Publishing House, New Delhi. Cotton Australia. Biotechnology and Cotton. https://cottonaustralia.com.au/fact-sheet (accessed on 14 October 2022). Cuozzo, M., O’Connell, K. M., Kaniewski, W., Fang, R. X., Chua, N. H., & Turner, N. E., (1988). Viral protection in transgenic tobacco plants expressing the cucumber mosaic virus coat protein or its antisense RNA. Bio/Technology, 6, 549–557. Davis, S. G., (2012). Constructing facts Bt cotton narratives in India. Economic and Political Weekly, 62–70.

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Dhillon, M., & Sharma, H. C., (2013). Comparative studies on the effects of Bt-transgenic and non-transgenic cotton on arthropod diversity. Journal of Environmental Biology, 34, 67–73. Dhillon, M., Gujar, G., & Kalia, V., (2011). Impact of Bt cotton on insect biodiversity in cotton ecosystem in India. Pak. Entomol., 33, 161–165. Dhurua, S., & Gujar, G. T., (2011). Field-evolved resistance to Bt toxin Cry1Ac in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), from India. Pest Manag. Sci., 67, 898–903. Dively, G. P., Venugopal, P. D., Bean, D., Whalen, J., Holmstrom, K., Kuhar, T. P., Doughty, H. B., et al., (2018). Regional pest suppression associated with widespread Bt maize adoption benefits vegetable growers. Proceedings of the National Academy of Sciences, 115, 3320–3325. Donovan, W. P., Rupar, M. J., Slaney, A. C., Malvar, T., Gawron-Burke, M. C., & Johnson, T. B., (1992). Characterization of two genes encoding Bacillus thuringiensis insecticidal crystal proteins toxic to coleoptera species. Appl. Environ. Microbiol., 58(12). Ekobu, M., Solera, M., Kyamanywa, S., Mwanga, R. O. M., Odongo, B., Ghislain, M., & Moar, W. J., (2010). Toxicity of seven Bacillus thuringiensis cry proteins against Cylas puncticollis and Cylas brunneus (Coleoptera: Brentidae) using a novel artificial diet. J. Econ. Entomol., 103, 1493–1502. EPA, (2000). Mammalian Toxicity Assessment Guidelines. https://archive.epa.gov/scipoly/ sap/meetings/web/pdf/finbtmamtox.pdf (accessed on 14 October 2022). Fabrick, J. A., Jech, L. F., & Henneberry, T. J., (2009). Novel pink bollworm resistance to the Bt toxin Cry1ac: Effects on mating, oviposition, larval development and survival. J. Insect Sci., 9. Fabrick, J. A., LeRoy, D. M., Unnithan, G. C., Yelich, A. J., Carrière, Y., Li, X., & Tabashnik, B. E., (2020). Shared and independent genetic basis of resistance to Bt toxin Cry2Ab in two strains of pink bollworm. Sci. Rep., 10. Fabrick, J. A., Mathew, L. G., LeRoy, D. M., Hull, J. J., Unnithan, G. C., Yelich, A. J., Carrière, Y., et al., (2020). Reduced cadherin expression associated with resistance to Bt toxin Cry1Ac in pink bollworm. Pest Manag. Sci., 76. Fabrick, J. A., Ponnuraj, J., Singh, A., Tanwar, R. K., & Unnithan, G. C., (2014). Alternative splicing and highly variable cadherin transcripts associated with field-evolved resistance of pink bollworm to Bt cotton in India. PLoS One, 9, e97900. Fabrick, J. A., Unnithan, G. C., Yelich, A. J., DeGain, B., Masson, L., Zhang, J., Carrière, Y., & Tabashnik, B. E., (2015). Multi-toxin resistance enables pink bollworm survival on pyramided Bt cotton. Sci. Rep., 5. Faisal, B. M., Farrukh, S. M., Wahid, M. A., Shakeel, A., & Maqbool, M., (2012). Adoption of Bt cotton: Threats and challenges. Chilean Journal of Agricultural Research, 72, 419. Federici, B. A., & Siegel, J. P., (2008). Safety Assessment of Bacillus Thuringiensis and Bt Crops Used in Insect Control (Vol. 172, p. 45). Food Science and Technology-New York-Marcel Dekker. Gandhi, V., & Namboodiri, N., (2009). Economics of BT Cotton Vis-a-Vis Non-Bt Cotton in India: A Study Across Four Major Cotton Growing States. Centre for Management in Agriculture, Indian Institute of Management, Ahmedabad. Gardner, M. L. G., (1988). Gastrointestinal absorption of intact proteins. Annu. Rev. Nutr., 8, 329–350.

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Luttrell, R. G., Ali, I., Allen, K. C., Young, S. Y. I., Szalanski, A., Williams, K., Lorenz, G., etal., (2004). Resistance to Bt in Arkansas populations of cotton bollworm. Proc. Beltwide Cott. Conf. Ma, H., Zhao, M., Wang, H., Wang, Z., Wang, Q., & Dong, H., (2014). Comparative incidence of cotton spider mites on transgenic Bt versus conventional cotton in relation to contents of secondary metabolites. Arthropod-Plant Interactions, 8, 1–7. Ma, W., & Zhang, T., (2019). Next-generation transgenic cotton: Pyramiding RNAi with Bt counters insect resistance. In: Methods in Molecular Biology (pp. 245–256). Humana Press Inc. Manjunath, T. M., (2012). Bt-cotton: It is Time to Stop the False Allegations! https:// www.dnaindia.com/analysis/comment-bt-cotton-it-is-time-to-stop-the-falseallegations-1671221 (accessed on 14 October 2022). Marques, L. H., Santos, A. C., Castro, B. A., Moscardini, V. F., Rosseto, J., Silva, O. A. B. N., & Babcock, J. M., (2019). Assessing the efficacy of Bacillus thuringiensis (Bt) pyramided proteins Cry1F, Cry1A.105, Cry2Ab2, and Vip3Aa20 expressed in Bt maize against lepidopteran pests in Brazil. J. Econ. Entomol., 112, 803–811. Mayee, C., Singh, P., Dongre, A., Rao, M., & Raj, S. (2011). Technical Bulletin from CICR No. 22. Critical Reviews in Biotechnology, 36(2), 317–326. Melo, A. L. D. A., Soccol, V. T., & Soccol, C. R., (2016). Bacillus thuringiensis: Mechanism of action, resistance, and new applications: A review. Critical Reviews in Biotechnology, 317–326. Mishra, R. R., (2020). Adoption of genetically modified crops can ensure food security in India. Natl. Acad. Sci. Lett., 43, 213–217. Mohan, S., et al. Integrated Pest Management for Cotton. https://www.niphm.gov.in/ IPMPackages/Cotton.pdf (accessed on 14 October 2022). Mohan, S., Monga, D., Kumar, R., Nagrare, V., Gokte-Narkhedkar, N., Vennila, S., Tanwar, R. K., Sharma, O. P., Bhagat, S., Agarwal, M., et al., (2014). Integrated Pest Management Package for Cotton, 84. Montagu, M. V., (1977). Vector for the introduction of NIF genes in plants? Genetic Engineering for Nitrogen Fixation, 159–179. Naik, V. C. B., Pusadkar, P. P., Waghmare, S. T., Raghavendra, K. P., Kranthi, S., Kumbhare, S., Nagrare, V. S., Kumar, R., Prabhulinga, T., Gokte-Narkhedkar, N., et al., (2020). Evidence for population expansion of cotton pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) in India. Sci. Rep., 10. Nair, R., Kamath, S. P., Mohan, K. S., Head, G., & Sumerford, D. V., (2016). Inheritance of field-relevant resistance to the Bacillus thuringiensis protein Cry1Ac in Pectinophora gossypiella (Lepidoptera: Gelechiidae) collected from India. Pest Manag. Sci., 72, 558–565. Nakamura, K., Oshie, K., Shimizu, M., Oeda, K., Ohkawa, H., & Takada, Y., (1990). Construction of chimeric insecticidal proteins between the 130-KDa and 135-KDa proteins of Bacillus thuringiensis Subsp. Aizawai for analysis of structure-function relationship. Agric. Biol. Chem., 54, 715–724. Naranjo, S. E., & Ellsworth, P. C., (2010). Fourteen years of Bt cotton advances IPM in Arizona. Southwest. Entomol., 35, 437–444. Naranjo, S. E., (2011). Impacts of Bt transgenic cotton on integrated pest management. J. Agric. Food Chem, 59, 5842–5851.

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Ni, M., Ma, W., Wang, X., Gao, M., Dai, Y., Wei, X., Zhang, L., Peng, Y., Chen, S., Ding, L., et al., (2017). Next-generation transgenic cotton: Pyramiding RNAi and Bt counters insect resistance. Plant Biotechnol. J., 15, 1204–1213. Ojha, A., Sree, K. S., Sachdev, B., Rashmi, M. A., Ravi, K. C., Suresh, P. J., Mohan, K. S., & Bhatnagar, R. K., (2014). Analysis of resistance to Cry1Ac in field-collected pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae), populations. GM Crops Food, 5, 280–286. Oppert, B., Morgan, T. D., & Kramer, K. J., (2011). Efficacy of Bacillus thuringiensis Cry3Aa protoxin and protease inhibitors against coleopteran storage pests. Pest Manag. Sci., 67, 568–573. Osman, G., Assaeedi, A. S. A., Organji, S., & El-Ghareeb, D. K., (2015). Bioinsecticide Bacillus thuringiensis a comprehensive review. Egyptian Journal of Biological Pest Control, 271. Pagano, M. C., (2014). Drought stress and mycorrhizal plant. In: Use of Microbes for the Alleviation of Soil Stresses (pp. 97–110). Springer New York. Pariza, M. W., & Foster, E. M., (1983). Determining the safety of enzymes used in food processing. Journal of Food Protection 46, 453–468. Pariza, M. W., & Johnson, E. A., (2001). Evaluating the safety of microbial enzyme preparations used in food processing: Update for a new century. Regulatory Toxicology and Pharmacology, 33, 173–186. Park, Y., Abdullah, M. A. F., Taylor, M. D., Rahman, K., & Adang, M. J., (2009). Enhancement of Bacillus thuringiensis Cry3Aa and Cry3Bb toxicities to coleopteran larvae by a toxin-binding fragment of an insect cadherin. Appl. Environ. Microbiol., 75, 3086–3092. Patton, D., (2014). China Launches Media Campaign to Back Genetically Modified Crops - Scientific American. https://www.scientificamerican.com/article/china-launchesmedia-campaign-to-back-genetically-modified-crops/ (accessed on 14 October 2022). Plata-Rueda, A., Quintero, H. A., Serrão, J. E., & Martínez, L. C., (2020). Insecticidal activity of Bacillus thuringiensis strains on the nettle caterpillar, euprosterna elaeasa (Lepidoptera: Limacodidae). Insects, 11, 310. Ponsard, S., Gutierrez, A. P., & Mills, N. J., (2002). Effect of Bt -toxin (Cry1Ac) in transgenic cotton on the adult longevity of four heteropteran predators. Environ. Entomol., 31, 1197–1205. Powell, C., (2015). How to Make a GMO - Science in the News, Harvard University, http:// sitn.hms.harvard.edu/flash/2015/how-to-make-a-gmo/ (accessed on 14 October 2022). Pray, C. E., Huang, J., Hu, R., & Rozelle, S., (2002). Five years of Bt cotton in China - the benefits continue. Plant J., 31, 423–430. Qaim, M., (2020). Bt cotton, yields and farmers’ benefits. Nature Plants. Nature Research, 1318–1319. Qaim, M., & De Janvry, A., (2005). Bt cotton and pesticide use in Argentina: Economic and environmental effects. Environ. Dev. Econ., 10, 179–200. Qiao, F., Huang, J., & Wang, X., (2017). Fifteen years of Bt cotton in China: Results from household surveys. World Dev., 98, 351–359. Rajendran, T. P., Birah, A., & Burange, P. S., (2018). Insect pests of cotton. In: Pests and Their Management (pp. 361–411). Springer Singapore.

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Rangel, G., (2015). From Corgis to Corn: A Brief Look at the Long History of GMO Technology. Science in the News, Harvard University, MA, USA. Raymond, P. J., McFarlane, I., Hartley, P. R., & Ceddia, G., (2011). The role of transgenic crops in sustainable development. Plant Biotechnol. J., 9, 2–21. Reed, J. P., & Halliday, W. R., (2001). Establishment of Cry9C susceptibility baselines for European corn borer and southwestern corn borer (Lepidoptera: Crambidae). J. Econ. Entomol., 94, 397–402. Ríos-Díez, J. D., Siegfried, B., & Saldamando-Benjumea, C. I., (2012). Susceptibility of Spodoptera frugiperda (Lepidoptera: Noctuidae) strains from central Colombia to Cry1Ab and Cry1Ac entotoxins of Bacillus thuringiensis. Southwest. Entomol., 37, 281–293. Rizzi, A., Raddadi, N., Sorlini, C., Nordgrd, L., Nielsen, K. M., & Da, D., (2012). The stability and degradation of dietary DNA in the gastrointestinal tract of mammals: Implications for horizontal gene transfer and the biosafety of GMOs. Critical Reviews in Food Science and Nutrition, 52, 142–161. Samir, M., & Abbas, T., (2018). Genetically engineered (modified) crops (Bacillus thuringiensis crops) and the world controversy on their safety. Egyptian Journal of Biological Pest Control, 28, 1–12. Sandeep, K. J., Jayaraj, J., Shanthi, M., Theradimani, M., Venkatasamy, B., Irulandi, S., & Prabhu, S., (2020). Potential of standard strains of Bacillus thuringiensis against the tomato pinworm, tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). Egypt. J. Biol. Pest Control, 30, 1–7. Sansinenea, E., (2012). Discovery and description of Bacillus thuringiensis. In: Bacillus Thuringiensis Biotechnology (PP. 3–18). Springer Netherlands. Schmidt, M. A., Lafayette, P. R., Artelt, B. A., & Parrott, W. A., (2008). A comparison of strategies for transformation with multiple genes via microprojectile-mediated bombardment. In: Vitro Cellular & Developmental Biology-Plant, 44, 162–168. Seiber, J. N., Coats, J., Duke, S. O., & Gross, A. D., (2014). Biopesticides: State of the art and future opportunities. Journal of Agricultural and Food Chemistry, 62, 11613–11619. Shudong, D., Jing, X., Qingwen, Z., Shiwen, Z., & Guanjun, X., (2003). Effect of transgenic Bt cotton on population dynamics of the non-target pests and natural enemies of pests. Kun Chong Xue Bao. Acta Entomol. Sin., 46, 1–5. Siegel, J. P., (2001). The mammalian safety of Bacillus thuringiensis-based insecticides. Journal of Invertebrate Pathology, 77, 13–21. Sisterson, M. S., Biggs, R. W., Olson, C., Carrière, Y., Dennehy, T. J., & Tabashnik, B. E., (2004). Arthropod abundance and diversity in Bt and non-Bt cotton fields. Environ. Entomol., 33(4), 921–929. Subramanian, A., & Qaim, M., (2009). Village-wide effects of agricultural biotechnology: The case of Bt cotton in India. World Dev., 37, 256–267. Tabashnik, B. E., Carrière, Y., & Gassmann, A., (2019). Global patterns of resistance to Bt crops highlighting pink bollworm in the United States, China, and India. Journal of Economic Entomology (pp. 2513–2523). Oxford University Press. Tabashnik, B. E., Liesner, L. R., Ellsworth, P. C., Unnithan, G. C., Fabrick, J. A., Naranjo, S. E., Li, X., Dennehy, T. J., Antilla, L., Staten, R. T., et al., (2020). Transgenic cotton and sterile insect releases synergize eradication of pink bollworm a century after it invaded the United States. Proc. Natl. Acad. Sci. U. S. A., 118.

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Tabashnik, B. E., Morin, S., Unnithan, G. C., Yelich, A. J., Ellers-Kirk, C., Harpold, V. S., Sisterson, M. S., Ellsworth, P. C., Dennehy, T. J., Antilla, L., et al., (2012). Sustained susceptibility of pink bollworm to Bt cotton in the United States. GM crops & food., 194–200. Veettil, C., Krishna, P., & Qaim, V. V., (2014). Bt cotton and ecosystem impacts of pesticide reductions. Global Food Discussion Papers. Vessey, J. K., (2002). First fruit: The creation of the flavr savr tomato and the birth of genetically engineered food. Choice Rev. Online, 39, 459–460. Vimala, D. P. S., Duraimurugan, P., Poorna, C. K. S. V., Vineela, V., & Hari, P. P., (2020). Novel formulations of Bacillus thuringiensis var. kurstaki: An eco-friendly approach for management of lepidopteran pests. World J. Microbiol. Biotechnol., 36, 1–14. Walters, F. S., Stacy, C. M., Mi, K. L., Palekar, N., & Chen, J. S., (2008). An engineered chymotrypsin/cathepsin G site in domain I renders Bacillus thuringiensis Cry3A active against western corn rootworm larvae. Appl. Environ. Microbiol., 74, 367–374. Wan, P., Huang, Y., Wu, H., Huang, M., Cong, S., Tabashnik, B. E., & Wu, K., (2012). Increased frequency of pink bollworm resistance to Bt toxin Cry1Ac in China. PLoS One, 7, e29975. Wan, P., Xu, D., Cong, S., Jiang, Y., Huang, Y., Wang, J., Wu, H., Wang, L., Wu, K., Carrière, Y., et al., (2017). Hybridizing transgenic Bt cotton with non-Bt cotton counters resistance in pink bollworm. Proc. Natl. Acad. Sci. U. S. A., 114, 5413–5418. Wang, L., Ma, Y., Wan, P., Liu, K., Xiao, Y., Wang, J., Cong, S., Xu, D., Wu, K., Fabrick, J. A., et al., (2018). Resistance to Bacillus thuringiensis linked with a cadherin transmembrane mutation affecting cellular trafficking in pink bollworm from China. Insect Biochem. Mol. Biol., 94, 28–35. Wang, L., Ma, Y., Wei, W., Wan, P., Liu, K., Xu, M., Cong, S., Wang, J., Xu, D., Xiao, Y., et al., (2020). Cadherin repeats 5 mutations associated with Bt resistance in a field-derived strain of pink bollworm. Sci. Reports, 10, 1–10. Wang, S., Just, D. R., & Pinstrup-Andersen, P., (2008). Bt-cotton and secondary pests. Int. J. Biotechnol., 10, 113–121. WHO. Environmental Health Criteria for Bacillus thuringiensis. https://www.who.int/ ipcs/publications/ehc/en/EHC217.PDF (accessed on 14 October 2022). Wilson, L., Downes, S., Khan, M., Whitehouse, M., Baker, G., Grundy, P., & Maas, S., (2013). IPM in the transgenic era: A review of the challenges from emerging pests in Australian cotton systems. Crop Pasture Sci., 64, 737. World Health Organization & International Programme on Chemical Safety. (‎1999)‎. Microbial pest control agent: Bacillus thuringiensis. World Health Organization. https:// apps.who.int/iris/handle/10665/42242Environmental Health Criteria, 217. Zeilinger, A. R., Olson, D. M., & Andow, D. A., (2016). Competitive release and outbreaks of non-target pests associated with transgenic Bt cotton. Ecol. Appl., 26, 1047–1054. Zhao, J. H., Ho, P., & Azadi, H., (2011). Benefits of Bt cotton counterbalanced by secondary pests? Perceptions of ecological change in China. Environ. Monit. Assess, 173, 985–994.

CHAPTER 3

Biohazards of Recombinant DNA Technology JOHRA KHAN1,2

Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Majmaah University, Majmaah, Saudi Arabia 1

Health and Basic Sciences Research Center, Majmaah University, Majmaah, Saudi Arabia

2

ABSTRACT The last three decades witnessed the development of rDNA and the production of genetically modified organisms (GMOs) and genetically modified microorganisms (GMM) and their application in various fields like; medicine, pharmacology, basic science, and agriculture. Their use on one side brought a revolution in agriculture and many other fields, but at the same time, it developed concerns on biohazards of these organisms. In this chapter, a brief review is provided concerning different biohazards and safety guidelines to use them as per the guideline of NIH and EPA. 3.1 INTRODUCTION Genetic engineering is a process of attaching or joining two different DNA molecules and then inserted them into the host organism for producing new genetic combinations that are beneficial in various fields such as medicine,

Genetic Engineering, Volume 2: Applications, Bioethics, and Biosafety. Tariq Ahmad Bhat & Jameel M. Al-Khayri (Eds.) © 2023 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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science, agriculture, industries, etc. (Mittler & Blumwald, 2010; Sandel, 2007). It is generally referred to as genetic engineering and gene-splicing. Recombinant DNA (rDNA) molecules are DNA molecules created by laboratory procedures or techniques of genetic recombination (Ceasar & Ignacimuthu, 2009). The sequence of DNA used for the creation of rDNA molecules can be derived from any species, such as human, fungal, plants, and bacterial (Radakovits et al., 2010). Additionally, DNA sequences that do not exist in nature can also be produced (Knott & Doudna, 2018). rDNA is the technique of taking genes from an organism and splicing them into the gene of another different type of organism which might result in alteration of the organism (Lütken et al., 2012). This process is mostly used to alter the phenotype of an organism (host) when a genetically modified (GM) vector is introduced and joined into the genome of an organism (Kappers et al., 2005; Kuzma, 2016). The increased use of GMOs and GMM in the environment can cause many biohazards, and it’s necessary to identify these hazards and understand the function of these organisms in a complex environment (Kittleson et al., 2012). NIH and USDA developed methods to handle issues related to biohazard and classified them based on the risk they pose (Hammer & Teklu, 2008). In this chapter, we discussed different types of risks and biohazards GMM, and GMO can cause. 3.2 BIOHAZARDS OF RECOMBINANT DNA TECHNOLOGY (RDT)

3.2.1 ASPECTS OF THE CONTROVERSY CONCERNING RECOMBINANT DNA (RDNA) The controversy concerning the use of rDNA technology started around the 1970s during research, Paul Berg refused to insert SV40 (monkey virus) to E. coli as this gene can turn a normal cell into a cancerous cell (Berg et al.). This research when conducted had helped to understand the nature of this lethal gene and the mechanism of cancer development. After the discovery of the restriction enzyme, pSC 101 plasmids came into existence which can be inserted into E. coli and development of rDNA (Gouw, 2018; Mohorčich & Reese, 2019). To investigate and observe the effect of rDNA technology a molecular biology committee was created in 1974

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(Studzinsky, 2012; Waddington et al., 2016). The main function of this committee was to impose a ban on certain rDNA experiments, in which we’re using some harmful microbes like animal tumor viruses, drugresistant bacteria, and other toxicity-generating microbes (Mohapatra & Ghosh, 2018; Wells, 2016). 3.2.1.1 GENETICALLY MODIFIED ORGANISMS (GMOS) AND THEIR BIOHAZARDS

Genetically modified organisms (GMOs) are a form of transgenic organisms with manipulated foreign genetic material using genetic engineering techniques (Małyska et al., 2014; Matveeva, 2016). These organisms are produced in the laboratory with desired characteristics that can be beneficial for both humans and animals. The most common techniques of modifications include the addition of beneficiary genes and the silencing of the harmful gene. GMOs are used in the production of food, experimental medicines, pharmaceutical drugs, and many other types of research (Lombardo & Zelasco, 2016; Phillips, 2008). GMOs were first created in 1973 as a bacterium with a gene of resistance against antibiotic kanamycin from other bacteria (Lee et al., 2009; Marmiroli, 2005). The world’s first transgenic animal was created in 1983 by Rudolf Jaenisch using a rat embryo. The first transgenic plant was developed by a combination of genetic engineering and tissue culture techniques. In this experiment, a tobacco plant was infected by agrobacterium, which can transfer resistant genes to plant cells. Using this plant cell by tissue culture technique, a new plant was developed having this resistant gene (Schouten et al., 2006). The biggest development in GMO production was the invention of the gene gun in 1987 (Liu et al., 2017; Nelson, 2012). It was first used in tobacco plants against Agrobacterium infection (Jacobsen & Schouten, 2009). 3.2.1.1.1 Biohazards With the development of GMOs, a lot of biosafety issues were raised which includes the effect of these GMOs on human health, the environment, and other organisms. Some of these hazards are:

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1. Toxicity: Corn developed using genetic engineering known as GM corn was found to express BT endotoxin Cry9C (Liu et al., 2016). A study conducted at the University of Caen, France on Bt-corn (Mon 810), was found to affect the viability of the human cell (Monnerat et al., 2015; Tabashnik & Gould, 2012). Some researchers concluded the effect only occurs in the presence of a higher concentration of toxins and is affected by the presence of other food components (Carrière et al., 2020; Siegfried & Hellmich, 2012). The introduction of toxin genes in plants can also cause modification in the structure of toxins that can cause a change in their selectivity (Al-Deeb & Wilde, 2005; Tarkalson et al., 2008). Smart Stax, is another variety of Bt-corn, which can produce 2 to 6 different toxins in higher concentration. To keep a check on the biohazards and their effect on the environment EPA (Environmental Protection Agency) of the USA accepted and registered 4 Bt-crops to be used for agriculture purpose from 1995 after rigorous research and reassessment of their potential effects on nature, non-targeted insects, and mammals including human. These crops are Bt-corn (Cry1Ab, and Cry1F), Bt potato (Cry3A), and Bt cotton (Cry1Ac) (Campagne et al., 2013; Tabashnik et al., 2009). The in vitro assessment of these Bt toxins was found to be unstable in the human gut and found to be degraded in the presence of digestive enzymes within 7 min of ingestion, but these studies do not confirm any toxic effect on the human cell. Another study on mice fed with 64 mg of toxin per kg of body weight of Bt rice floor for 90 days did not cause any effect on their liver, intestine, kidney, and blood tissues (Tian et al., 2014; Yu et al., 2011). A study by Hall (2011) concerning food from Bt crops and human health (Yang et al., 2016), found little effect of these toxins on human health (Abbas, 2018; Heckel, 2012). According to him, our diets are very complex, and it is difficult to trace the effect of toxins in acute or chronic form by Bt toxins. To measure any direct or indirect effect of food items like the meat of animals that were raised on transgenic crops will take decades to confirm the result of Bt toxins (Abbas, 2018; Saker et al., 2011). To analyze the effect of Bt toxins on 60 biochemical parameters, a study was conducted on rats’blood and organs, based on serum and urine analysis for 14 weeks were recorded. Bt crop feed rates were

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compared with non-Bt crop feed rats in a group of 6. The analysis of liver and kidney samples revealed the effect of toxins on the tissues of these organs, and also on the heart, spleen, glands, and hematopoietic system. The study of Vendomis et al. (2009) was refused by the French High Council of Biotechnologies Scientific Committee, saying it shows no evidence of toxicity by Bt-maize (De Vendômois et al., 2009). Another study focused on the effect of Bt toxin (Cry1Ab1) on pregnant, non-pregnant, and fetal blood was analyzed and recorded in 0.19 to 0.30% concentration in 93% pregnant and 80% fetal blood samples (Abbas, 2018). As most studies were concerned with the transfer of Bt toxin DNA in human gut bacteria, in 2004 a study was conducted on human volunteers on a diet using GM soybean (Kılıç & Akay, 2008; Verma et al., 2011). This study resulted in the negative presence of rDNA in human gut bacteria. Another study funded by the European Arm of Greenpeace found a minor risk of Bt-toxins on rats’ liver, however, the European Food Safety Authority did not consider any biologically significant risk of these toxins on human health (Bawa & Anilakumar, 2013; Coupe & Capel, 2016; Dona & Arvanitoyannis, 2009). Many other studies conducted on sheep and pigs fed with Bt-maize and cotton plant for three months recorded no significant change in the histology of kidney and liver tissues (Dona & Arvanitoyannis, 2009). To analyze the presence of Bt-toxins in processed food (Chougule & Bonning, 2012), different types of Cry protein were introduced to heat-stable studies similar to the food processing industrial environment (Han et al., 2009; Then & Bauer-Panskus, 2017). After heat processing, all Cry proteins broke and lost their insecticidal activity, and no data to this day in any study found these toxins affecting biological functioning or hematological changes in any tissue was recorded (Koch et al., 2015; Park et al., 2014). 2. Allergenicity by Bt-Crops: After toxicity, the other big concern on using Bt-crops are the incidences of increasing allergic reactions recorded in many countries (Goodman et al., 2016; Then, 2010). The first incident of allergic reaction came to notice when Brazilian nut protein was transferred to a soybean plant to increase its nutritional value started producing allergies in human

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volunteers (Bawa & Anilakumar, 2013). This occurred due to the transfer of allergenic protein transfer in GM soybean (BertóMoran et al., 2013; Sanden et al., 2004), and as its identification was possible early this crop has never been approved for market distribution (Mathesius et al., 2009). The second incidence of allergy was noted during research on a bean plant recombination to develop resistance against weevil but on feeding it to mice developed an immune response in lungs, because of which the crop was destroyed and never send for production (Kim et al., 2006; Świątkiewicz et al., 2010). Allergies due to GM soybean in UK and US market and started the controversy of biohazards due to GM crops (Hoff et al., 2007). The alternative measure for using GM crops can be safety testing of GM crops and their product before releasing them in marked or exposing them to the general population (Zhu et al., 2004). The allergies that develop in our immune system is only possible when a protein can bind with IgE (Ladics et al., 2014), so more exposure of GM protein to people can cause more allergies but only if the person has corresponding sensitivity (Geng et al., 2015; Pearce et al., 2010). Recombinant technology to develop GM crops not always causes to form new proteins but due to deletion of some gene, some proteins are also lost, and this removal can also cause allergies. Identification of these allergic proteins and their removal can be an alternative to use GM crops as its ongoing in the case of soybean and peanut (Panda et al., 2013; Tiwari et al., 2008). 3. Impact of GMO on Environment: Just growing GM crops does not cause any harm to the farming environment but some related practices harm the environment. Growing GM crops leads to the use of one type of insecticide and herbicides again and again for a long time that causes the eradication of some wild plants growing around the farm (Bawa & Anilakumar, 2013; Coupe & Capel, 2016). The loss of these wild plants not only loses plant varieties but also the gene bank of the environment. In 1999 a large-scale study was conducted in farms of the UK to identify the effect of herbicide-tolerant GM crops (Carvalho, 2006; Thrall et al., 2011). This study continued till 2006, and the results of the study showed that reduction in weeds also reduces the number of insects feeding

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on them. This is the loss of biodiversity and damage of wildlife (Raman, 2017; Thakore, 2006). This issue can be controlled by the rotation of crops and the introduction of insect-resistant GM crop development. The growing of GM crops has another issue is there crossbreeding with wild plants (Herman et al., 2019). This crossbreeding between wild and GM crops will risk the health of other wild varieties. These crossbreeding can also cause the selection of wild or weedy characters that make these plants wilder and the transfer of resistant genes can cause the development of herbicideresistant weed growth (Chapman & Burke, 2006). These changes can also cause the loss of many genes from the natural gene pool that are useful in the selection of important characters (Strive & Cox, 2019). 3.2.2 EFFECT OF GM ON NON-TARGETED ORGANISMS GM crops are developed with rDNA technology either with new nutritional additions or to develop resistance against herbicides or insecticides, but some non-target organisms are also affected by these GM crops (Potts et al., 2016). Based on their function, these non-targeted organisms are classified as: (i) herbivore animals feeding on crops and weeds; (ii) pollinators like bees and butterflies; (iii) endangered species; and (iv) soil flora. The herbivore animals that feed on GM crops transfer the effect of toxins to the food chain which will affect other plants and animals directly or indirectly (Burgio et al., 2011; Verma et al., 2011). Poultry animals feeding on Bt maize were exposed to Cry34Aba and Cry35Ab1 toxins whereas the freshwater dolphins were also found to be exposed to these toxins, no noticeable effect was recorded, whereas Cry1Ab toxin of Bt maize was found to produce no effect the soil flora like bacteria, fungi, nematodes, protozoa, and earthworms (Craig et al., 2008; Tsatsakis et al., 2017). The pollen protein present in Bt maize (Cry3A, Cry2Ab1, Cry3Bb1, Cry1Ab, Cry9C, Cry34Ab, and Cry35Ab1) was found to have no effect larvae or adults of honeybee (Li et al., 2008). But if these toxins are ingested in high concentration larvae of monarch butterfly were affected and their population size also decreases. Big animals and humans were not found affected by the concentration of these toxins (Hendriksma et al., 2011; Perry et al., 2010).

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3.3 GENETICALLY MODIFIED MICROORGANISMS (GMMS) AND RELATED BIOHAZARDS Over the last 3 decades after the development of GMOs, genetically modified microorganisms (GMMs) that brought a revolution in the field of medicine, pharmaceuticals, agriculture, and other bioscience research areas. With these developments, many safety concerns also need to be addressed. GMMs include bacteria, fungi, viruses, and another plasmodium with recombinant genetic material (Janse et al., 2011; Rasala & Mayfield, 2015). The release of GMMs, when released into the environment can adversely affect human health and the environment by producing irreversible effects. The issues related to GMM can be addressed by studying the behavior of these organisms in a controlled complex environment followed by data analysis for risk assessment (Snow et al., 2005; Tibayrenc & Ayala, 2012). 3.3.1 BIOHAZARDS OF GMM RELEASED IN THE ENVIRONMENT The release of recombinant viruses in the form of used vaccines is evaluated through SIFER summary information format for environment release which evaluates the impact of viruses that contain rDNA are analyzed for: (i) potential effect of an organism that may establish in the environment; (ii) the distribution of organism in different organs; and (iii) plan to control if contingency problem occurs (Aneja, 2007; Lin et al., 2016). An example of this issue came to light when a woman got infected with a GM vaccine virus after her pet in Ohio. There is a Pest control Measure to be taken before it is released in mammals, especially about its species susceptibility, as species with a broad host range should never be used for pest control (Beyene et al., 2018; Kuck et al., 2018). Outside a lab when a pathogen is used for pest control directly or indirectly it enters the environment and is released in that area. In 1953, a non-GM myxoma virus-infected rabbit was released in the local community to control the population of rabbits in that area (Elsworth et al., 2014). This infection spread in Europe causing devastating destruction in rabbit farms in Europe causing big economic damage (Foldvari et al., 2016; Pannier & Shea, 2004). GMM analysis for impact on environment need to analyze microbial ecology, as there is no standard protocol has been established (Masaki et al., 2010; Oh et al., 2019). In another study, Pseudomonas fluorescens CHAO was

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used to change soil bacterial population around cucumber roots. A GMM Pseudomonas putida was developed as an insecticide; its use was found to produce an effect on fungal species which are non-targeted by GMM (Beyene et al., 2018; Lee et al., 2017). A similar effect was recorded with GM baculovirus which was found to produce an effect on the aquatic microbial community of the surrounding area. Researchers dealing with GMM considered the viability of recombinant microorganisms are less in comparison to indigenous species of bacteria in a complex environment (Nutter, 2006; Parsyan et al., 2006). The study by Velkov explained that the DNA of GMM can result in increasing the viability of the microorganism due to differences in cellular processes, adaptive process, resistance due to activation of cellular responses, and activation of her mutagenic process (Olaitan et al., 2014). Some studies on GMM also recorded that these recombinant organisms can survive up to six years in an environment without a potential host by plasmid transfer (de Souza, 2016; Marraffini & Sontheimer, 2010). For Example, GM fungi with a stable chromosomal structure were found to with contacting weed control agents but at the same time, too much exposure to GM fungi and increased host range in an uncontrolled environment can cause a potential hazard to the environment (AlvarezOrdóñez et al., 2015; Ibraheem & Ndimba, 2013). So, during the release of GMM pre-evaluation should be done to determine the rate or possibility of horizontal DNA transfer (Saini et al., 2018; Salvestrini et al., 2019). In bacterial species, the exchange of DNA between distant species and even between gram-positive and gram-negative bacteria is well recorded (Kerr et al., 2013; Shimizu, 2014). Some bacterial species are identified that are capable of genetic material exchange in natural condition and based on this capacity, they are classified into three groups as shown in Figure 3.1. As these transfers are not considered safe and can cause biohazard in an environment so it is necessary to take precautions. Undesirable gene transfer between GMM and wild type can lead to leather novel combination, as reported in the case of GM Pseudomonas DNA, rhizosphere, and spermosphere (Naamala et al., 2016; Weaver & Morris, 2005). These DNA transfers can be reduced by performing rDNA in the chromosome in place of plasmids. The frequency of DNA transfer increases if GMM organisms provide an advantage to the host, as in bacteriophage which provides virulence factor, additional substrate spectrum, and resistance to antibiotics (Clough, 2020; Raaijmakers & Mazzola, 2012; Shmaefsky, 2010).

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FIGURE 3.1 transfer.

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Different groups showing members who are capable of horizontal DNA

3.4 PUBLIC AND GOVERNMENTAL CONCERNS RELATED TO BIOHAZARDS OF GMMS To control the biohazards from GMMs, EPA in the U.S. published “Biotechnological Program Under Toxic Substances Control Act,” defined GMM in commercial industries as intergeneric and new organisms and according to this act any industry that wants to use or produce GMM should provide a document “Microbial Commercial Activity Notice,” 90 days before the use of the organism. EPA needs to evaluate the impact of GMMs release in the environment (Al-Jassim & Hong, 2017). Around 60 days before the field test, the experimental release application “Biotechnological Program Under Toxic Substances Control Act,” must be submitted. NIH (National Institute of Health) registered strict guidelines for using recombinant organisms under the Office of rDNA activities (Goujon, 2019; Milewski, 2019; Weiner, 2020). NIH directs to work directly with institutions handling the GMM both at the physical and biological level

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(Gostin et al., 2014; Gottweis, 2005; Kamely, 2013). NIH classifies the risk groups as given in Figure 3.2.

FIGURE 3.2

Different risk groups classified by NIH for risk assessment.

The safety regulations of USDA for GMM are focused on recombinant microbes, especially plant pathogens. USDA allows the use of these microbes by APHIS (Animal and Plant Health Inspection Services) (Council, 2004). There are two types of permits provided by APHIS: (i) GMMs that require field testing of the potential plant pathogen; and (ii) GMMs that are required to bring these pathogens to United States (Council, 2004). Both these permits must be documented, including details of the organism, source, and type of gene used for developing GMMs, reason for study, method, and design of the study, and step by step procedure to disseminate these organisms at the site of testing. 3.5 CONCLUSION rDNA technology allows the formation of new DNA artificially from incorporating two or more single molecules of genetic material. The

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different branches of DNA modification include genetic engineering, rDNA technology, CRISPR, and gene splicing are used for direct gene manipulation of a microbe. With the emergence of these new methods, the productions of a large amount of artificial proteins useful in the medical field are produced in large quantity for their use in pharmaceutical industries. These large extracellular proteins are very important for chronic replacement therapies or for the cure of deadly disease conditions. GMM and GMO are increasing day by day. A lot of disagreements are also noticed in relation to the safety issues of these GM crops and their labeling as GMO products and GMM. Environmental risk assessment is a structural approach used to analyze the risk associated with GM crops. The final results of these changes in DNA may not be limited to some specific characteristics of the substituted gene. Therefore, it is important to safeguard the use and release of these organisms in nature so, they do not produce any risk to damage to the environment or to human health. KEYWORDS • • • • • •

Bt-cotton cry protein environmental risk assessment genetically modified microorganisms recombinant DNA toxicity

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frugiperda on transgenic Bt cotton: Implications for resistance management. Scientific Reports, 6(1), 1–7. Yu, H. L., Li, Y. H., & Wu, K. M., (2011). Risk assessment and ecological effects of transgenic Bacillus thuringiensis crops on non‐target organisms F. Journal of Integrative Plant Biology, 53(7), 520–538. Zhu, Y., Li, D., Wang, F., Yin, J., & Jin, H., (2004). Nutritional assessment and fate of DNA of soybean meal from roundup ready or conventional soybeans using rats. Archives of Animal Nutrition, 58(4), 295–310.

CHAPTER 4

Genetic Engineering and Human Welfare MOHD. SULIMAN DAR1 and SHUGUFTA RASHEED2

Department of Botany Government Degree College, Anantnag, Jammu and Kashmir, India 1

CBT, Department of Botany, University of Kashmir, Srinagar, Jammu and Kashmir, India

2

ABSTRACT Genetic engineering has proved a promising field in the welfare of human survival. It involves manipulation or modification of genomic structure either by removing or introducing the DNA fragments that can alter the gene expression with altered function in target organisms. These alterations can be achieved either by using bacteriophages, or plasmids or by direct microinjections. The importunity of foreign DAN with its required design is very important in the successful outcome of genetic engineering. It is through the key improvements in the sequencing of DNAthat the alterations, additions, insertions and deletions were made possible. With its successful advent and results genetic engineering has revolutionized the fields like medicine, industry, horticulture, agriculture, research and industry. Every single area of molecular biology has witnessed a tremendous advancement through the procedures of genetic engineering and the organisms so designed are known as genetically modified organisms (GMOs). After the first small-scale trials of GM crops in 1990, the acceptance and adoption

Genetic Engineering, Volume 2: Applications, Bioethics, and Biosafety. Tariq Ahmad Bhat & Jameel M. Al-Khayri (Eds.) © 2023 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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of these crops have increased considerably. Despite the increased yield and enhanced financial benefits, the technology has resulted in disagreement and a source of controversy and despite the expert intervention and consensus, the safety of GM foods still remains a leading concern with opponents. The source of foreign traits in GMOs is very diverse, it can be a bacterium or a distant wild relative of an organism. These foreign genes can be introduced into the target organisms by numerous techniques like recombinant DNA, gene delivery, electroporation or gene editing which encompass a series of steps using numerous sophisticated genetic engineering tools. One of the promising outcomes of genetic engineering is the restoration of the environment by remediation of deteriorated ecosystems and the development of eco-friendly processes and procedures for the reduction of environmental pollution. Despite its promising and luring outcomes, it has raised certain concerns like welfare and ethical issues, the emergence of resistant pathogenic strains and other possible threats which need to be seriously considered. 4.1 INTRODUCTION Genomic structural modification or manipulation of an organism, either by introducing or removing DNA is known as genetic engineering. It involves the various procedures for the planned management of genetic material to augment, modify, or repair arrangement or role of genetic material. Genetic engineering techniques that are settled near the end of the 20th century encompass the chemical intertwining of diverse strands of DNA commonly using either plasmids or bacteriophages, or by straight microinjection. It involves the list of various techniques that can transform the cells genetically (Robert & Baylis, 2008). The genomic structure of the cells is altered by transference of genes within and across precincts of species resulting in the production of novel organisms. It involves the use of recombinant DNA (rDNA) technology which involves straight use of one or many genes of an organism. Usually, a foreign gene introduced in an organism gives it a wanted expression. This ability to alter the genotype of an organism was made possible by key advancements in DNA sequencing, understanding of cellular physiology and development of cloning vehicles (vectors). The design, delivery, and persistence of foreign DNA (transgenic DNA) are the important components on which genetic engineering mostly

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relies. Thus, the marketable industrial transformation of living organisms into novel and profitable life forms is the result of this technique of genetic engineering (Fox, 1988). Genetic engineering involves the series of steps like gene separation, amendment, exclusion, and finally recombination. Genetic engineering has a lot to do with human welfare. It has revolutionized numerous fields like industry, agriculture, research, and medicine. Genetic engineering has made it possible to correct genetic disorders through gene treatment, understanding of gene function and manifestation through GMOs, production of drugs by the creation of model animals and enzymes for industrial purposes. In spite of several benefits and possibilities that this new technology ensures, in the background hides many ethical questions and possible threats that need to be seriously considered. If not taken into care by all stakeholders like implementing agencies of industry and government, academicians, supporters of eco-welfare organizations and the public at large, this promising technology may prove worse and will not only disturb our lives but also the generations to follow. The vast majority of crop species are produced by humans using orthodox breeding techniques on wild plants. These methods of plant breeding depend on selective and judicious breeding procedures. Conservative breeding procedures, although simple and ingenuous to perform, have the applied result of altering an organism’s genotype, thus producing new characters (Fisher, 2021). 4.2 GENETIC ENGINEERING The recent advances in cell and molecular biology have initiated the hope of the emergence of new technology that can be used to improve or correct the harmful genotypes at the DNA level. This method of correcting defective genes in an organism by molecular techniques or simulated amalgamation of required genes and their consequential relocation into the genetic structure of an organism created a new discipline called genetic engineering. Genetic engineering can be used to knock out or remove genes which are then inserted randomly or targeted at places of interest in the genome. The artifact that is obtained by this technique is known as recombinant DNA (rDNA). This procedure can be used to detach and duplicate single copy of a gene or a segment of DNA into an unlimited number of identical copies. Vectors like plasmids and phages have made this possible because they have the ability to replicate in a host (e.g., in

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E. coli) in a manner as natural even after loaded with external DNA. This foreign DNA replicates loyally with the parent DNA. This method is used to isolate, clone, and characterize the genes of interest and is called gene cloning. This procedure has resulted in considerable advancement in every single area of molecular biology. The organisms created through this technique are considered to be genetically altered and are known as genetically modified organisms (GMOs). It takes about 10 years on average to produce new varieties of crop through genetic engineering. With time, the application of genetic engineering using recombinant DNA technology (RDT) augmented and the first small scale field trials of GMOs were carried out in USA and Canada in 1990, followed by the first marketable release of GM crops in 1992. After that, acceptance of GM plants by agriculturalists has enlarged considerably. Despite the profits harvested from genetically designed crop varieties, there has been widespread disapproval of this technology from ethics considerations, environmental perspectives, and enhanced concern of common masses with a monopoly of corporate control of crop varieties (Wieczorek & Wright, 2012). The increased use of genetically improved crops has delivered enhanced financial benefits to farmers in numerous countries where they were used. At the same time, the technology has also resulted in the disagreement and a source of controversy. It is this controversy that led to the destruction of early field trials by anti-GM activists. Although the expert consensus which made it clear that the food derived from GM crops causes no potential threat to human health than straight food, GM food safety still remains a leading apprehension with opponents. The potential issues raised include intellectual property rights (IPRs) and control of food supply, gene drift and impact on non-target entities. These apprehensions, which led to an international treaty of development of a regulatory framework in 1975, resulted in the Cartagena Protocol on Biosafety, adopted in 2000. Member countries have established their own regulatory systems concerning GMOs, with the remarkable differences present between Europe and the USA. The prospective increase in crop yields, improvement of agricultural practices or additional nutritional quality of products is only because of foreign or new traits introduced into the crop plants by genetic engineering. For example, weedicide resistant transgenic crop plants allow farmers to spray weedicides without damaging the crop plants and affecting the yield (Datta, 2013). Similarly, soil erosion and water loss can be prevented, and

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tillage can be reduced by using herbicide-tolerant crops by farmers. Insecticidal toxins expressed in transgenic plants can resist attacks from insects. Crops altered to ward off insects are a substitute to insecticide sprays and are also cost-effective. The use of synthetic insecticides is reduced which may not reach to all parts of the plant. The nutritional quality of food can be enhanced by genetically designing crops for example, “golden rice” has been engineered and designed to produce beta-carotene. In the near future, edible vaccines, can be produced from plants by the use of technology of genetic engineering. Characters and traits expressed in genetically altered plants are obtained from diverse sources. A gene from the bacterium Salmonella has been used to destroy the pesticide glyphosphate (Roundup TM) in cultivars of soybeans, corn, canola, and cotton. From the bacterium Bacillus thuringiensis (Bt), a gene responsible for insecticidal toxin have been introduced in transgenic corn, potato, and cotton plants. Production of vitamin A in golden rice is due to the gene obtained from the Erwinia uredovora (bacterium); other traits in golden rice are due to the genes derived from the Narcissus. Golden rice which embodies several new traits in it involves the introduction of many genes from diverse sources into a rice plant. These genes offer a multistep biochemical pathway required for the expression of new chemicals and products. Common rice grain, which forms a staple food for much of the world population, lacks vitamin A. Due to the vitamin A deficiency, an estimated 100 million to 200 million children worldwide are suffering from a condition of blindness: escalated susceptibility to diarrhea, respiratory infection, and childhood diseases, such as measles. Beta-carotene and other carotenes (pigments, red, yellow, and orange found in carrots and other vegetables) from the precursors of vitamin A. Beta-carotene is synthesized in the chloroplasts of rice plant but not in the edible seed tissue. Ingo Potrykus et al. found a precursor to carotenoid production geranylgeranyl diphosphate (GGPP), present in rice seed. They altered genetically rice to golden rice to express the enzymes compulsory for the alteration of GGPP to beta-carotene. Four biochemical reactions are required to synthesize beta-carotene, from geranylgeranyl diphosphate (GGPP), each catalyzed by a different enzyme. The genes necessary for the complete biochemical pathway for beta-carotene production were introduced into rice using all the three plasmids present in Agrobacterium tumefaciens (Priyadarshan, 2019). Only three enzymes were used instead of four as the bacterial enzyme phytoene desaturase achieves

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what two enzymes (phytoene desaturase and beta-carotene desaturase) in plants do. In developing nations due to the limited facility of refrigeration, transportation, and disposable needle supplies, edible vaccines available in crops could be of immense help. The gene that encodes an E. coli protein has been expressed in potatoes by Hugh Mason et al. (Boyce Thompson Institute). The new, modified potatoes developed antibodies to the protein when eaten raw, and investigation is started to see whether the antibodies will defend against diarrhea caused by disease-causing E. coli. Paul Berg in 1972 constructed the first rDNA and in the same year gene transfer experiments were performed by Herbert Boyer and Stanley Cohen. First time in the history of genetics, Rudolf Jaenisch (1974) had created genetically altered mice. Following this successful intervention, genetically altered or genetically planned tobacco plant species were developed in 1976. From 1960 to 1990, restriction digestion, ligation of DNA segments, and PCR like techniques were settled, which strengthened the field of this new technology. In 1982, an antibiotic-resistant tobacco plant was produced by genetically modifying the genome of tobacco plant (Shetty et al., 2018). The first field trials of genetically modified (GM) herbicide-resistant tobacco were carried out in USA and France in 1986. Marc Van Montagu and Jeff Schell were the first to find a company known as Plant Genetic System in 1987 in Belgium to develop insect resistant (tobacco) plants by integrating genes from Bt that produced insecticidal proteins. China in 1992 was the first country to allow commercialization of transgenic plants, including a virus-resistant tobacco, which was later on withdrawn from the market in 1997. Flavr Savr tomato was the first genetically altered crop permitted for sale in the U.S., in 1994. The tomato was hard, took longer to soften after ripening, thereby enhancing shelf life. Tobacco was the first genetically engineered crop resilient to the bromoxynil, an herbicide permitted by the EU in 1994 and was marketed in Europe. In 1995 Bt potato was the first pesticide producing crop approved by the US Environmental Protection Agency (US-EPA). Canola with altered and manipulated oil composition (developed by Calgene), bromoxynilresistant cotton, Bt cotton (developed by Monsanto), Bt maize (developed by Ciba-Geigy), glyphosphate-resistant soyabeans (developed by Monsanto), virus-resistant squash (developed by Asgrow) and delayed ripening tomatoes (developed by DNAP, Zeneca/Peto and Monsanto) were permitted in 1995 (Raman, 2017). About 35 authorizations had been made in the mid of last half of the 20th century to commercially grow 8

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genetically altered food crops and an ornamental plant (carnation) with 8 different traits incorporated in them in EU and other 6 countries. Early In 2000, golden rice enriched with vitamin-A was developed though as of 2016 it was not yet in commercial production. Robert Fraley, Marc Van Montagu, and Mary-Dell Chilton the leaders of three research teams were the first to apply genetic engineering to crops in 1013. These leaders were awarded the World Food Prize for improving the quality, quantity, and availability of food in the world. 4.3 TYPE OF TECHNIQUES USED IN GENETIC ENGINEERING 4.3.1 RECOMBINANT DNA (RDNA)

In this type of genomic engineering technology, an artificial molecule of DNA is constructed by ligating two different DNAs from different sources using physical methods. This technology known as recombinant DNA technology (RDT) involves the insertion of gene of interest into the plasmid vector for gene transfer experiments. 4.3.2 GENE DELIVERING This technique known as gene delivering technique involves the insertion of gene of interest into the host genome. 4.3.3 ELECTROPORATION It is a highly efficient method for introducing external genes into the tissue culture cells. Viral vector-mediated gene transfer, liposome-mediated gene transfer, transposon-mediated gene transfer is some of the methods used for that. 4.3.4 GENE EDITING TECHNIQUE Editing of the genome of interest is done by this technique. It involves the removal of undesired DNA sequences or insertion of new genes into the host genome. Some known gene editing tools used in genetic engineering experiments are CRISPR-CAS9, ZFN, and TALEN.

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4.4 STEPS INVOLVED IN GENE TRANSFER Gene transfer in genetic engineering involves a series of steps which are discussed in subsections. 4.4.1 ISOLATION, PURIFICATION, AND IDENTIFICATION OF THE GENE OR DNA SEGMENT THAT IS TO BE TRANSFERRED The genes that are to be manipulated are either isolated or synthesized artificially for transformation leading to the production of transgenic plants and animals. Significant progress has been made in the techniques for the isolation of a variety of genes. With the help of RDT or cloning any gene from any organism can be isolated as full genomes of organisms are sequenced. One of the best and for easier techniques of gene isolation is polymerase chain reaction (PCR) (Kaufman & Nixon, 1996). It is a powerful tool of gene amplification that can amplify a given gene sequence, the sequences hence produced can be isolated by using the technique of gel electrophoresis. PCR has become a staple tool in every genetic engineering laboratory. Some of the other strategies used for isolation and purification of genes are transposon tagging; use of DNA or RNA probes, subtractive hybridization, differential screening, map-based cloning, use of RFLP maps or chromosome mapping and use of chromosome jumping. During last 30 years considerable achievement has been made in the isolation, purification, and characterization of genes. Many novel genes for human welfare have been obtained. Purification and characterization of gene from Collybia velutipes for oxalate decarboxylase (Mehta & Datta, 1991) and molecular gene cloning of gene from Amaranthus that encodes seed-specific protein nutritionally balanced amino acid structure (Raina & Datta, 1992) are the examples of isolation of novel genes from early 90s. 4.4.2 TRANSFER OF ISOLATED AND PURIFIED GENE INTO THE TARGET GENOME BY USING CLONING VEHICLES OR VECTORS When the gene is incorporated into vector, promoter, terminator, and marker genes are also added to it. To express efficiently further modifications are also done. The foreign gene is inserted usually into the embryonic

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stem cells in case of animals while as in plants it can be inserted into any tissue that can be cultured into fully developed plant. 4.4.3 SELECTION AND EXPRESSION OF INTRODUCED GENES

The recombinant construct is lastly incorporated into a suitable host cell or organism. This process can be accomplished in numerous ways and is called transfection (for eukaryotes) or transformation (for prokaryotes). It is the final step where the expression of transgenes is manipulated and controlled. Selection based on antibiotics is usually used to declare which cells have successfully incorporated the external recombinant construct. Since only the plasmid vector contains the required antibiotic resistance gene, so only the transgenic cells will grow in the presence of antibiotics. Once isolated and purified, transgenic cells can hopefully start expressing the desired character encoded by the gene of interest (https://ib.bioninja. com.au/standard-level/topic-3-genetics/35-genetic-modification-and/ gene-transfer.html). 4.5 GENETIC ENGINEERING TOOLS The following tools are involved in genetic engineering: 1. Polymerase Chain Reaction (PCR): The process of producing and replicating multiple copies of required genes is known as polymerase chain reaction (PCR). The manipulation of DNA replication in the laboratory was made possible by the discovery of thermostable DNA polymerases, such as Taq Polymerase. This technique is used to amplify quantities of DNA segments. With the help of primers gene of interest is identified and then replicated to the required interest. The amplified genes are then isolated and purified using gel electrophoresis. 2. Restriction Enzymes (Molecular Scissor): With the discovery of restriction endonucleases known as molecular scissors the protein and genetic engineering has become possible. These enzymes are specific and based on the nucleotide sequence, cut DNA at specific locations. Many smaller DNA fragments of varying sizes with sticky ends are produced by these restriction enzymes (REs).

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These fragments can be separated using gel electrophoresis or chromatography. 3. Gel Electrophoresis: The DNA after extracted using PCR and REs can be visualized in terms of size and type by gel electrophoresis. Because purifying DAN from cell culture by using REs and PCR wouldn’t be of much use if we couldn’t visualize it. It is also used to detect DNA inserts and knockouts. 4. DNA Ligases: Ligases are the enzymes that help in union. These enzymes form covalent bonds between nucleotide chains and help in binding. This leads to the formation of recombinant strands or closure of circular strands that have been cut by REs. The filling of gaps and phosphorylation of 5’-ends of DNA strands is done by DNA polymerase 1 and polynucleotide kinase respectively. 5. Plasmids: Small circular segments of DNA which have independent existence outside the bacterial genome are known as plasmids. They are capable of independent and self-replication. They are used as cloning vehicles to carry genes across cells and microorganisms. The required genes after isolation, purification, and amplification by PCR are loaded on plasmid by cutting gene and plasmid using REs and are then ligated together. The resulting product is known as recombinant DNA (rDNA) molecule. Cosmids which are combined products of plasmids and bacteriophage genes can also be used as vectors or cloning vehicles. 6. Transformation/Transduction: The process of transferring genes on a vector like plasmid, into the host or target cell is called transformation. Electroporation which makes the host cells competent or temporarily permeable to vector exposes the cells to an environmental change. The efficiency of plasmid to behave as vector depends upon the size, larger the size of the plasmid lowers the efficiency with which it is taken up by the host cell. Larger DNA segments are more easily cloned using bacteriophage, retrovirus, or other viral vectors or cosmids in a method called Transduction. In regenerative medicine, phage or viral vectors are used, but such introduction may cause the unwanted insertion of DNA in parts of our chromosomes, causing complications and even cancer.

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7. Identifying Transgenic Organisms: Whether the cell has taken the foreign DNA or not during transformation has to be identified because all cells do not take up DNA during transformation. Therefore, the cells that undergo transformation and those that do not must be identified and separate from each other. Generally, antibiotic resistance is used as an indicator, and transgenic cells can be selected based on the expression of those genes and their ability to grow on media containing antibiotics. The other methods of identifying depend on the presence of some other indicator proteins such as the x-gal/lacZ system. Also, green fluorescence protein allows the identification based on color and fluorescence. 4.6 BENEFITS OF GENETIC ENGINEERING FOR HUMAN WELFARE Genetic engineering encompasses immense agricultural and industrial takeoff. It has a role in crop improvement, medicine, genetic research, production, and designing of therapeutic drugs and food industry. Development of GMOs became possible by its intervention, which led to the development of GM foods. Today markets are flooded with best quality fruits and vegetables and their quantities have been increased manifold and qualities improved. 4.6.1 FOOD INDUSTRY By applying genetic engineering know how the genetic structure of GM foods have been changed utilizing genes from different plant sources, particularly from their wild relatives. Quality traits for an ideal attribute are searched and isolated from one plant or organism and are embedded into another plant or cell for its improvement. Transmissible changed nourishments are acquired from hereditarily adjusted organisms, or transgenic crops. Hereditary adjustments have led to the hereditary building of various improved traits in transgenic crops. In short GM foods are tastier, more nutritious, and cost-effective with enhanced shelf life. They are more advanced in color and size wherever this trait is required. On the other hand, GM crops are disease and drought-resistant that requires fewer environmental resources (water and fertilizer) for their growth. These crops are also immune to the insect infestation.

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4.6.2 MEDICINE Genetic engineering has been extensively exploited in mass production of insulin, follistim (FSH for rewarding infertility), human development hormones (GH), monoclonal antibodies, human albumin, vaccines, antihemophilic variables and many other different medications. Gene pharming involves the production of pharmaceuticals using genetically engineered plants and animals. It is a convenient substitute to traditional pharmaceutical development because genetically engineered organisms (GEOs) are relatively inexpensive to produce and maintain. Large quantities of pharmaceuticals, such as vaccines, enzymes, hormones, and antibodies can be produced from pharmed organisms. Drugs of various human diseases can be produced from recombinant proteins by pharming. Different diseases and deficiencies in patients can be successfully treated by direct injection of these therapeutic products into their bodies. Gene therapy and vaccine production has made a remarkable realization to overcome consequences of various critical and pandemic diseases. This all breakthrough in medicinal fabrication became possible because of genetic engineering. 4.6.3 APPLICATION OF GENETIC ENGINEERING IN ENVIRONMENT Restoration of the environment by the use of genetically altered microorganisms, plants, and animals is currently a promising field of genetic engineering. Development of microorganisms and biocatalysts for remediation of deteriorated and contaminated ecosystems is actively done by utilizing genetic engineering. Also, development of eco-friendly processes and procedures such as producing recombinant strains of organisms for bio-fuel production have a visible effect on the reduction of environmental pollution. Greenhouse effect and global warming are directly linked to increased level of carbon dioxide emission in the atmosphere. So, to reduce the concentration of carbon dioxide in the atmosphere, efforts are made to limit its production consequent emission into the atmosphere. In this background, the enzyme ribulose biphosphate carboxylase (RuBPCase) which is closely associated with CO2 fixation is being designed in a fashion which results in augmented photosynthetic activity leading to the increased fixation of carbon dioxide. Genetically altered organisms

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are used in cleaning up of oil spills in oceans which is a major threat to aquatic ecosystems. New strains of micro-algae like mutants of Anacystis nidulans and Oocystis spp. are being developed which can tolerate high concentrations of CO2. 4.6.4 CROP IMPROVEMENT AND TRANSGENESIS

A large number of useful compounds like metabolites and therapeutic products have been obtained from plants designed and altered genetically. Transgenic crops with multiple traits like disease resistance, herbicide resistance, insect resistance, drought and salt resistance have been fabricated. Transgenic plants have ensured better quality and quantity of crops grown in the fields. 4.6.5 ANIMAL IMPROVEMENT AND TRANSGENESIS Acquaintance of foreign gene into an animal to improve it in attributes is known as animal transgenesis. Transgenic animals in this way at long express the attribute of the required quality. These animals are furthermore made to anticipate the capability of various qualities to create legitimate treatment of diseases. These transgenics are used as models for testing the safety of vaccines before they are injected into humans. Medicines to treat diseases like phenylketonuria and hereditary emphysema are expected from transgenic animals. Human proteins can be obtained from the milk of genetically altered cows. 4.7 SHORTCOMINGS OF GENETIC ENGINEERING Since its advent several moral and ethical issues associated with the use of genetically engineered products and gene therapy has been raised. Generally, the nutritional value of a food product is compromised in order to suffice its economic value. New resistant pathogenic strains can get evolved which may be very difficult to control onwards. Because of its adverse effects, concerns like invasiveness of procedures, requirement of large number of organisms, welfare issues, and emergence of new resistant pathogenic strains appear to be much visible and are to be controlled and

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tackled carefully. Also, the side effects of gene therapy and the use of viruses in it can prove harmful hither to unnoticed. The technology though marvelous is not cost-effective as gene therapy costs may go as high as 60,000 USD or even more. KEYWORDS • • • • • •

electroporation gene editing genetic engineering genetically modified organisms recombinant DNA traits

REFERENCES Datta, A., (2013). Genetic engineering for improving quality and productivity of crops. Agriculture & Food Security, 2, 15. Fisher, M. R., (2021). Biotechnology and Genetic Engineering. https://chem.libretexts. org/@go/page/14660 (accessed on 14 October 2022). Fox, M. W., (1988). Genetic engineering biotechnology: Animal welfare and environmental concerns. Applied Animal Behavior Science, 20, 83–94. https://ib.bioninja.com.au/standard-level/topic-3-genetics/35-genetic-modification-and/ gene-transfer.html (accessed on 14 October 2022). Kaufman, R. I., & Nixon, B. T., (1996). Use of PCR to isolate genes encoding σ54-dependent activators from diverse bacteria. J. Bacteriol., 178, 3967–3970. Mehta, A., & Datta, A., (1991). Oxalate decarboxylase from Collybia velutipes. Purification, characterization, and cDNA cloning. Journal of Biological Chemistry, 266, 23548–23553. Priyadarshan, P. M., (2019). Genetic engineering. In: Plant Breeding: Classical to Modern. Springer, Singapore. Raina, A., & Datta, A., (1992). Molecular cloning of a gene encoding a seed-specific protein with nutritionally balanced amino acid composition from Amaranthus. Proc Natl Acad Sci, USA, 89, 11774–11778. Raman, R., (2017). The impact of genetically modified (GM) crops in modern agriculture: A review. GM Crops Food, 8, 195–208.

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Robert, J. S., & Baylis, F., (2008). In: Harald, K., & Kris, H., (eds.), Genetic Engineering (pp. 35–39). International Encyclopedia of Public Health, Academic Press. Shetty, M. J., Chandan, K. C., Krishna, H. C., & Aparna, G. S., (2018). Genetically modified crops: An overview. Journal of Pharmacognosy and Phytochemistry, 7, 2405–2410. Wieczorek, A. M., & Wright, M. G., (2012). History of agricultural biotechnology: How crop development has evolved. Nature Education Knowledge, 3, 9.

CHAPTER 5

Genetically Modified Organisms: Scope and Challenges

MUHAMMAD ISHTIAQ, MUBASHIR MAZHAR, MEHWISH MAQBOOL, and MUHAMMAD WAQAS Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur, Azad Jammu and Kashmir (AJK), Pakistan

ABSTRACT Food security and quality products are inevitable for life sustenance. The population of human being is exponentially increasing, and this is creating food issues. There is one way to increase the cultivation area of agriculture or increase double crops cultivation in a year. For this new crop cultivars have to be produced, and this can be comprehended through using the latest biotechnological techniques. For this genetic engineering technique (GET) is very promising to cope this plethora of food security. The plants or other organisms produced through GET is currently being promulgated and organisms produced using this GET are called genetically modified organisms (GMOs). Albeit these GMOs have mitigated the issue of food scarcity, but it has also many other challenges of side effects with different paradigms. There is an issue to reveal and analyze biodiversity loss, and other health issues of GMOs. There is need to study the endogenous toxins in plants and/or other GMOs. This chapter encompasses the holistic preview of GMOs, their merits, and demerits. GMOs are those which have foreign genes. Genes with desired characteristics are identified and isolated. Then these isolated genes are Genetic Engineering, Volume 2: Applications, Bioethics, and Biosafety. Tariq Ahmad Bhat & Jameel M. Al-Khayri (Eds.) © 2023 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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introduced into target organisms with the help of biological vectors. These transgenic organisms having genes of interest from other organisms incorporated into their genome are called GMOs. GMOs are essentially to be analyzed properly prior to use. The key features in context of use of GMOs may exhibit side-effects for health. The use positive use of GMOs is also beneficial to cope the needs of everlasting increment in the human world population. The GMOs are providing very promising scope in human history. 5.1 INTRODUCTION With advancement in biotechnological protocols, scientists are able to detect particular genes controlling a specific characteristic. Isolation of genes of interest is also possible and at the same time transfer of genes of interest to new organisms is now possible. The organisms which contain genes of interest isolated from other organisms are termed as genetically modified organisms (GMOs) (James, 2016). Biotechnology has unable scientists to alter the genetic makeup of organisms and to make them an ideal organisms with desired characteristics. Such GMOs are widely being prepared to meet human needs (Hill, 2005). GMOs are not only good yielding and less laborious to handle but they come up with the ability to control the pollution, pests, and abiotic stresses of heavy metals. GMOs may be used to produce specific proteins or growth promoters for other specified organisms, including human beings. Some of the genetically modified microorganism have the ability of reclamation of acidic soils (Sehubent, 2002). The introduction of GMOs still needs careful observations as they are able to disturb the ecological cycles or lifecycles of other species found nearby (Tzotzos et al., 2010). GMOs are no doubt able to give good results in agriculture, medicine, and other fields of human interest; however to introduce a GMO in the environment has strict regulations both from the local governmental bodies as well as from the international regulatory. Yet it is need of hour that much scrutiny of GMOs must be carefully done. Every nation has a responsibility to develop certain regulations and laws to safeguard the interests of the masses (Kelper & Davies, 2010). GMOs are better colonizers as compared to wild types. It is seen that they dominate in an area when grown uncontrolled. It may have severe ecological consequences (Johnson et al., 2007). Genetic modification may also alter the working

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of genes other than foreign genes, and it can be disastrous for ecosystems (EFSA, 2006). 5.2 SCOPE OF GENETICALLY MODIFIED ORGANISMS (GMOS) 5.2.1 BETTER YIELD AND IMPROVED PRODUCTIVITY

With logarithmic increase in human population, the world needs more food to feed this large number of people. On the other hand, agricultural fields are continuously being destroyed for the construction of houses, industries, and parks. In this situation it is desperately needed that farmers could get more yield in less area of agricultural field. Scientists have made GM crops which have better yield and need less capital for irrigation, fertilizers, and herbicides or pesticides (Remi et al., 2010). Developing countries are provided with these GM crop seeds to boost their production and get more income to sustain their economy (Hall et al., 2013). GM crops have developed resistance from pests and farmers are not required to spray with pesticides, GM crops are also able to withstand certain environmental stresses with great efficiency. All these characteristics combine to give good production at the end of harvesting season. It was never possible previously with conventional methods of agriculture (Baulcombe et al., 2013). With no use of herbicides and pesticides, farmer get benefit in terms of capital, and the environment gets no pollution. About 37% of decrease in use of pesticides was studied with 21% increase in the yield of same GM crops (Klumper & Qasim, 2014). Although GM crops have boosted the production of edibles yet a huge amount of grains and fodder is required to sustain the food supply (Mofa et al., 2015). 5.2.2 INTRODUCTION OF ABIOTIC RESISTANCE Environmental stresses including drought, water logging, salinity, and severe winds destroy the crops hence yield output is greatly decreased (Foolad, 2007). Unawareness and poor town planning leads to problem of municipal wastes and lack of proper drainage. Wastewater from households is drained in some agricultural fields near the towns, which causes the salt levels to increase beyond the sustainable limits and hence crops grown in such fields yield very low (Dorothen & Ramanjula, 2005).

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Scientist have prepared GM tomatoes that can tolerate a wide range of salinity. Such GMOs have better transport systems of salts with greater efficiency. Arabidopsis was studied for such modifications, and ultimately, genes of interest for better elimination of sodium and protons were entered in tomatoes which were grown in saline fields and yet managed to get good yields (Apse et al., 2009). Later these tomatoes were studied for their nutritional aspects, and it was found that fruits that are edible were safe for consumption and only leaves flooded with greater amounts of sodium (Zhang & Blumwald, 2001). Drought is also a great reducer in production of crops. Scientists have found FRI gene is capable to cope drought with modification in transpiration levels. These genes have been successfully introduced in wheat, maize, and mettle crops and it was found that these crops were easily able to withstand drought (Chandler & Tauka, 2018). Likewise, HVA1 genes were isolated from barley and Agrobacterium-mediated transfer protocol was followed to introduce this gene in mulberry. GM mulberry was found to be effectively produced in drought as well as in saline environment (Checker et al., 2012). 5.2.3 INTRODUCTION OF BIOTIC RESISTANCE GM crops with better resistance to environmental stresses showed better growth and development which was ultimately ended up with better yield. So, scientists also identified the genes responsible for resistance to biotic stresses, including pests and herbicides in the case of crop plants (Nowicki et al., 2013). Plants with resistance to a number of pests, bacteria, and fungi have been prepared (Kikulwe et al., 2008). By the introduction of pests’ resistance genes isolated from Bacillus thuringiensis (Bt) cotton and maize were able to withstand a variety of pests and pathogens (Velimirov et al., 2008). Biotechnology has made it possible to achieve greater yields from GM plants (Askari & Pessarkali, 2008). Bt genes introduced in plants resulted in the development of great resistance in tobacco from hornworm, in tomato from Keiferia lycorersicella, Heliothis zea and Helicoverpa armigera (Kumar, 2004; Chan et al., 2010). With advancement in biotechnological protocols GM plants have been prepared which can be easily propagated and it is very likely to decrease the harvesting cost, herbicides cost, and better productivity is seen. Chrysanthemum plants have been modified genetically which can resist aphid

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attack hence more caffeine can be achieved as yield from less agricultural land and low consumption of credit (Kim et al., 2011). 5.2.4 BETTER STORAGE AND SHELF LIVES GMOs are so modified that to enhance their after-harvest life. Genes responsible for delayed ripening of fruits and delayed senescence were identified and introduced in crops to increase their shelf lives and to reduce huge losses due to after harvest molding of cash crops in developing countries where there were no proper means of disinfectants. Tomatoes were made able to ripen well after their natural fruit ripening rhythm (Alexander & Grierson, 2002). Tomatoes were modified to retain their flavor for a long period after harvesting and these model GMOs were very first to be licensed in 1994 under the name of flavor saver tomatoes (Hall et al., 2013). Polygalacturonase is an enzyme that is produced which softens the flesh and skin of tomatoes hence their flavor is lost. Naturally wild type tomatoes can be stored for only 15 days with good taste and texture. Production of this enzyme was suppressed in GM tomatoes and such fruits were able to retain their taste and texture for more than 45 days. Antisense techniques for silencing ethylene producing genes were adopted to produce climacteric plants which were able to produce fruits with longer shelf life (Buncombe, 2010). 5.2.5 IMPROVEMENT IN ATTRACTION Floriculture has also been revolutionized by introducing newer traits in flowers which have made these flowers more attractive as compared to wild types (Clark et al., 2002) it has also made it possible to get more flowers in less time from a number of ornamental plants (Jiang et al., 2010). Traditional cross-fertilization techniques were not able to introduce new traits in Orchids. Genetic modification has achieved a milestone in the production of very fascinating flower colors and patterns (Dasilva et al., 2011). Mostly rose flowers were pink and red which were the appealing colors for large community however some people as in China and Japan demand blue color because they think blue color shows more purity of love and is attractive as per esthetic point of view. For the production of blue color in rose flowers a gene dolphinin was identified and introduced

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by the help of Agrobacterium-mediated genetic transfer. These GM roses produced blue flowers in Japan and Australia (Chandler & Tauaka, 2018). 5.2.6 NUTRIENT-RICH PRODUCTIVITY GMOs are available which produce nutrient-rich fruits or leaves that are easy to digest, are glutton free and nutrients are easily absorbed from such foods. GMOs with better food size, texture, color, and flavor have been produced (Murno, 2019). A specific gene that code for the production of phytoene desaturase, was identified and introduced in tomatoes which have more quantity of pro-vitamin A. Such fruits have the same quantities of carotenoids too as compared to wild type (Chandler & Tauaka, 2018). Anthocyanin was responsible for enhancement in the production of antioxidants. Isoflavone quantities were enhanced by introducing special transcription factors that were isolated from Arabidopsis thaliana it is well known anticancer agent. Isoflavone from soyabean were successfully introduced in tomatoes (Nayor, 2011). Modified tomatoes were rich in antioxidants and helped to treat cancers (Mike, 2008). Banana with enhanced production of pro-vitamin A were produced which were used to overcome the malnutrition in people of Africa, especially malnourished children (Ortiz & Swannen, 2014). GM banana was rich in iron and provitamin A along with better nutrients (Ginlio, 2012). GM tomatoes were also produced which were 25 times richer in pro-vitamin A and carotenes similarly bananas with genetic modifications were sufficiently rich in nutrients to overcome the problem of malnutrition in children of Uganda (Askari et al., 2018). 5.2.7 PRODUCTION OF ANTIGENS AND OTHER BIOPHARMACEUTICALS Bananas, lettuce, and tomatoes with altered genes were able to produce special antigens which were helpful in increasing the immune responses and good mental health. Nutritional analysis of these GM crops showed that they were perfectly safe for human consumption (Aukita et al., 2016). GM crops were able to prepare human antigens responsible for delayed aging and helpful to reduce the pathogenic attacks. Such antigens

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producing fruits were edible and safe. These vaccines are affordable, safe, and more effective as compared to traditional vaccines (Horn et al., 2004). Recently number of other pharmaceutics have been prepared by GM crops which are cheaper and easier to achieve immunity against certain pathogens (Fischer et al., 2004). Developing countries are continuously adopting plant-oriented vaccines because of their cost-effective and easy to manage production (Giddings et al., 2000). Human growth hormone was produced by GM crops in 1986 since then, scientists have achieved several milestones in the production of useful hormones and antigens (Liete et al., 2000). Hepatitis B Virus vaccine has been produced by entero-toxigenic Escherichia coli (E. coli). Proteins useful in tissue replacement and special protein called spider silk polymers that are largely used in surgery for wound healing are being produced from GM plants (Ma et al., 2003). 5.2.8 BIODEGRADATION AND ECOLOGICAL GAINS GMOs are helpful to recycle certain organic as well as inorganic wastes. Microorganisms that are commonly found in decaying logs have special genes responsible for the production of digesting enzymes like catalase, cellulose, decarboxylases, and many others. Genes for these enzymes are identified and introduced into some bacteria that are able to reclaim acidic soil and also capable of degradation of plastics (Tzotzos et al., 2010). 5.3 CHALLENGES WITH GENETICALLY MODIFIED ORGANISMS (GMOS) 5.3.1 ENVIRONMENTAL CHALLENGES GMOs are need of hour to ensure food availability but at the same time, it is highly important to maintain human and environmental health at sustainable levels (Robent & Guillerme, 2014). If GMOs are not carefully regulated then ecological issues of invasive weeds, unintended propagation of certain species and disappearance of some species may be resulted. GMOs may be responsible for the elimination of certain genes to other wild types which in turn may be disastrous. Genes may also move to sexually related species without knowing the consequences (Clark et al., 2006). So, for the application of GMOs regulations and clear-cut principles should be

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followed. It must also be regularly monitored to that minimize the possible negative effects. Safety measures should be strict and small violations may result in disapproval of GM crops for cultivation (EFSA, 2011). 5.3.2 ECOLOGICAL CHALLENGES Most of the time, genetic alterations are made to benefit human being, but it is likely to produce negative effects on other organisms, which are known as NTOs. Foreign genes inserted into transgenic organisms may be useful for humans, but it may cause disturbances to other organisms in their life cycles or livability (Devos et al., 2009). Some of the species get devastating effects by application of GM crops. Bee’s population around the globe is tremendously decreasing due to usage of GM crops (Holmes, 2010). Some of the pathogens may also arise due to uncontrolled propagation of GM crops. Pathogenic bluetongue virus (BTV) is a striking example that has spread in European states. BTV spread has raised several questions, and almost all of the answers are related to the use of excessive GMOs (Jones et al., 2008). Some of the other health issues in Europe have roots in GMOs propagation that is why some of the European states including Italy have banned cultivation of GM crops and certain states have not allowed scientists to even grow GM crops designed for research purposes (Wilkinson & Ford, 2007). 5.3.3 SOCIAL CHALLENGES In the near future, people will be more adapted to biotechnology and GM crops, but it is still a great deal for ignorant people or people living in remote area where there is no awareness campaign. Every country has tried to convince its population and to sort out their concerns; however, it is estimated that it will take 10 to 15 years to change the concept of an ordinary people towards the use of GM crops and animals (Jones et al., 2008). It is believed by a large community that GMOs will change the natural outflow and it is against the laws of nature. People resist in application of GMOs due to fear of God or nature and they think approval of GMOs will certainly have consequences (Fischer et al., 2004). One such challenge is to eradicate the superstitious thoughts. GM producers have solved this issue by labeling the GM products and have

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given a choice to all consumers whether they choose GM products or natural ones (Dasilva et al., 2011). In India several court decisions have been noted to stop the cultivation of GM crops due to possible negative environmental effects. Farmers have also recorded protests for the use of such modified organisms considering it against the laws of nature (Nowicki et al., 2013). GM products have faced great criticism in the United Kingdom (UK) despite the fact that GM products have better nutritional values as compared to wild types. Most of the products that were genetically modified (GM) or raised from GMOs were largely not accepted by UK citizens. It was due to European Union laws and regulations as well as citizens own choice (Dibden et al., 2013). EU states have banned a number of GM products due to possible health, ecological or environmental issue related to their usage. Australia and USA have largely adopted GM products in almost all the forms. Citizens of the USA and Australia have concerned our use of GMOs however governments have labeled GM products and sorted out the confusion (Sinenins, 2008). 5.3.4 ECONOMIC CHALLENGES There is clear-cut evidence that GMOs are better in several traits however it may give rise to market monopoly of certain companies that own the patent of GMOs. If a GM crop or product is largely accepted by any community or territory private company will charge huge amounts for purchase of this product. Consequently, it is estimated that private companies’ outcomes will be manifold, but farmers will not be able to get benefited much (Demont et al., 2007). For the cultivation of GM crops, farmers have to buy their certified seeds from any company or organization that owns the rights to sell these seeds in any particular area. Farmers have to buy from only these sellers which is also a type of monopoly. Such sellers charge doubles the amount described or settled by government (Beckmann et al., 2006; Moghissi et al., 2018). Due to large community and corrupt officials, it is difficult to regulate the GMO laws. Seeds are also not properly labeled, and transportation is very slow due to all these factors farmers did not get theoretical yields, so they do not grow GM crops regularly. Several such cases have been reported in India, Pakistan, and China, e.g., GM eggplant, mustard, and Bt

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rice were not able to get approval of a large community of poor farmers (Pingali, 2018). Due to lack of credit, capital, and experts developing countries face difficulties in application, approval, and regulation of GM crops (Chann et al., 2010). 5.3.5 ETHICAL CHALLENGES Numerous ethical challenges are related to GMOs application. It is thought to change the ability, quality, and nature of wild types (GTEC, 2006). Bt cotton in USA gave defected bolls which were deformed with less strength. GM soybeans with dormant seeds were produced. Later it was noted that dormant soybeans have a greater amount of lignin in their seed coats (Crag et al., 2008). GMOs can also affect the production of other species in the field. It may have unwanted effects on the ecosystem (Wilkinson & Ford, 2007). Release of GM animals will alter the community structure and have consequences on GM animals too which is ethically not acceptable (Oberhouser et al., 2001). Transgenic organisms with better traits may suppress their counterparts in nature (Moghissi et al., 2018). Incorporated genes may have an effect on mating and the integrity of other wild types may be harmed in some ways (Pleasantly et al., 2001). Genes may be incorporated into species related to GMOs and may affect their natural habit, habitat, lifecycle, and metabolism (Dively et al., 2014). 5.3.6 GENE POOL DISTURBANCE For the production of GMOs, one or a few genes of desired traits are isolated and inserted into transgenic organism with the help of vectors. Research has shown that genes are linked with one another, and their expression is also related to one another. As one gene is inserted in GMO it can change the whole genome of that organism and this variation may have defective expression leading to fatal effects (Ho, 2008). As isolated genes are transported with the help of vectors, these vectors have other genes for antibiotic resistance, terminator genes and also promoter genes. These genes are also transferred along with the gene of interest and alter the genetic makeup of GMO. With such alterations, some genes may have increased expression while other genes expression may be decreased,

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which can be disastrous not only to GMO but also to the generations after it (Checker et al., 2012; Crag et al., 2008; Haigh, 2004). One such example is promoter genes of cauliflower mosaic virus (CaMV) were associated in almost all GMOs in which CaMV was used as a vector. Scientists believe that this promoter gene if activated in receptors may have consequences on human and other animal health (Pingali, 2018; Lai, 2012). 5.3.7 HEALTH CHALLENGES A most important factor is human health and while application of GM crops or animals it is certainly kept in mind. Available research shows concerns about human health. Much research in this regard is desperately needed to eradicate the negative effects of GMOs. Antibiotic resistance and metabolic disorders are major issues related to human health (Holmes, 2010). Studies on model organism have shown increased senility and immature sperm production by consumption of GM crops. Albino rats were used to study the possible effects of GM crops (potato and soya), and it was found that it has harmed some of the organism and some of the rats died. Cotton seeds that were GM showed abnormal fetal growth, infertility in males and premature births (Velimirov et al., 2008). GM cereals were found to be adversely affecting the citizen’s health in the UK, Australia, and the USA. Individuals showed allergic responses while using GM products (Ho, 2008). In South Asia, there are several studies that have shown severe allergic responses and nutritional disorders by consumption of GM fruit, especially farmers that were indulged in handling and harvesting of Bt cotton were found to develop skin and respiratory allergies (Kurungenti, 2008). In China and Japan, Bt cotton was seen to produce a special protein that was responsible for skin allergy (Pasini et al., 2002). 5.3.8 REGULATORY CHALLENGES GMOs are helpful to get more yield and with better characteristics as compared to wild types. Studies have also shown concerns about human health, social, ethical, and economical aspects. Governments and local bodies need to carefully administer GMOs and must strictly follow guidelines issued by regulatory departments. It is therefore their responsibility to regulate the application and utilization of GMOs. Studies have shown

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that people require proper labeling of GM products so that if it is not acceptable by consumers, they can choose from other products rather than GM products (Daniel, 2004). Prices of GM products should also be regulated to avoid monopolies. Surveys have resulted that a large community is not satisfied with regulations and availability of GM products. A large number of farmers said they were not interested in GM seed due to their sky-high prices. So, it is needed that GM products must be carefully regulated. In remote area several GM products have never reached due to logistic issues (Carter & Gurere, 2003). 5.3.9 UNINTENDED IMPACTS ON OTHER SPECIES GMOs may have toxic effects on other species and their population may decline, resulting in loss of ecosystem. For example, Bt maize with Cry1 Ab genes have a toxic effect on monarch larvae, so in this field, monarch butterfly was eliminated (Jhonson et al., 2007; Dively et al., 2014). Toxicological studies showed that monarch butterfly is a great vector for pollens from anther to stigma and due to a decrease in their population production of non-target crops was greatly decreased (Pleasant et al., 2001). GM maize fields were able to withstand a large number of pests, but pollination was much decreased in other crops nearby due to a decrease in monarch butterfly. It is noted that the monarch butterfly population has declined to 0.6% in the USA, where Bt maize has been regularly grown (Wolt et al., 2003). Study of Bt cotton that was conducted to study effect of Bt genes expression on insects showed that some of the insects were completely eliminated from the study fields and due to eradication of some of the species other species population was bloomed (Sears et al., 2001). Bt genes expression was studied in corn, and it was found that GM corn leaves were not good for the health of farm animals when it was used as fodder (Jesse & Obrycki, 2000). 5.3.10 METAPHYSICAL AND SPIRITUAL CHALLENGES Application of GMOs has some religious and philosophical concerns also. Some groups in developing countries see it as “God’s Play” and are strictly

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against the use of GMOs. Some people think transfer of special genes from plants into animals and vice versa is illogical and immoral. A large group of religious people think it is against the laws of nature and God. They think people should restrain from GM products as nature or God with curse the users of these products (Hoban, 2004). 5.3.11 CHALLENGING FUTURE PROSPECTIVE

Scientists believe that genes of an organism work in collaboration and when any foreign gene is introduced into genome of any organism it will change whole genome. With this changes in genome working of some other genes may also be changed. So, it is necessary to study GMOs for almost 4 to 5 generations (Nielsen & Townsend, 2004). Due to the introduction of new traits in an organism, it can flow to related species, and this flow may have devastating effects on other species that were not targeted in scientific protocols (Chan et al., 2010). 5.4 CONCLUSION GMOs are helpful to give better productivity and enhanced yield. It is possible to reduce the usage of herbicides and pesticides by using GM crops with biotic and abiotic resistance. GM fruits and vegetables can be stored for a longer time. GM flowers have more attractive attributes like color, fragrance, shape, and texture. GM plants yield specific antigens, proteins, vitamins, and vaccines. GMOs may be used for bioremediation and biodegradation as well as reclamation of acidic soils. Although GMOs come with desired genes for desired products yet there are several challenges related to their approval and application. There are environmental challenges of GMOs that their application will disturb biogeochemical cycles leading to pollution. GMOs can dominate in an ecosystem resulting in the disappearance of other species. GM seeds and products are responsible for economic monopolies. There are social, moral, ethical, philosophical, and religious concerns with the application of GMOs. GM products may have adverse effects on human health or the health of other organisms. Population explosion is a huge problem which can lead to a shortage of food in the near future. GMOs are to be considered to overcome this shortage. GMOs are helpful to improve the

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agricultural yield, growth, nutritional values and biotic as well as abiotic resistance can be easily enhanced in GMOs. GM crops are easy to grow, with no or lesser need of fertilizers. GM crops can yield more with shorter time span. But there are ecological, economic, environmental, and ethical concerns related to the application of GMOs. GMOs should be adopted to enhance the production and desired characteristics, but their approval must follow strict regulations and their application should be carefully managed and monitored. KEYWORDS • • • • • •

abiotic resistance bioremediation biotechnical advances biotechnology genetically modified organisms population explosion

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Phillips, P. W., & McNeill, H., (2000). Labeling for GM foods: Theory and practice. AgBioForum., 3(4), 219–224. Pidgeon, J. D., May, M. J., Perry, J. N., & Poppy, G. M., (2007). Mitigation of indirect environmental effects of GM crops. Proceedings of the Royal Society B: Biological Sciences, 274(1617), 1475–1479. Pingali, P., (2018). Will The Gene Revolution Reach the Poor? Lessons from the Green Revolution. Paper presented at the Wageningen University. Mansholt. Wageningen, The Netherlands. Pleasants, J. M., Hellmich, R. L., Dively, G. P., et al., (2001). Corn pollen deposition on milkweeds in and near cornfields. Proceedings of the National Academy of Sciences of the United States of America, 98(21), 11919–11924. Remi, A., (2010). Agricultural biotechnology: Does it work in Africa? In: GMOs for African Agriculture: Challenges and Opportunities. Workshop proceedings report. Academy of Science of South Africa. Robert, P., & Guillermo, H., (2014). Food Safety: State-of-Play, Current and Future Challenges. In-depth analysis. Schubert, D., (2002). A different perspective on GM food. Nature Biotechnology, 20(10), 969. Sears, M., et al., (2001). Impact of Bt corn on monarch butterfly populations: A risk assessment. Proceedings of the National Academy of Sciences, 98, 11937–11942. Sinemus K. (2008). Communicating science, technical, ethical and social aspects related to GMOs: in 1st Global Conference on GMO Analysis. Villa Erba, Como, Italy 24–27 June 2008. Available from: http://file.cbd.int/database/attachment/?id=2330. Tzotzos, G. T., Head, G. P., & Hull, R., (2010). Principles of risk assessment. Genetically Modified Plants, 2009, 33–63. Velimirov, A., Binter, C., & Zentek, J., (2008). Biological Effects of Transgenic Maize NK603xMON810 Fed in Long Term Reproduction Studies in Mice. Report by Institute fur Ernahrung, Austria. Wilkinson, M. J., & Ford, C. S., (2007). Estimating the potential for ecological harm from gene flow to crop wild relatives. Collection of Biosafety Reviews, 3, 42–47. Wolt, J. D., Peterson, R. K., Bystrak, P., & Meade, T., (2003). A screening level approach for non-target insect risk assessment: Transgenic Bt corn pollen and the monarch butterfly (Lepidoptera: Danaiidae). Environmental Entomology, 32, 237–247. Zhang, H. X., & Blumwald, E., (2001). Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat. Biotechnol., 19(8), 765–768.

CHAPTER 6

Genetic Engineering of Horticultural Crops

MUHAMMAD ISHTIAQ, MUBASHIR MAZHAR, MEHWISH MAQBOOL, and MAHNOOR MUZAMIL Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur, Azad Jammu and Kashmir (AJK), Pakistan

ABSTRACT The relationship of man with plants is since time immemorial and plants provide all fundamental necessities of life. The plants particularly horticulturally grown plants are of high worth because these are screened and man-selected plants to meet their pivotal needs. The horticultural plants are cultivated for coping the needs or for earning livelihood by selling these in local or international markets. Horticulture crops many needs of life and also revenue of life. But due to traditional methods, the yield and productivity has been reduced to low or minimum form. The current era is time of genetic engineering which is technique to induct the desired character in the plant of interest with inserting gene of action through latest biotechnological techniques. The application of genetic engineering and DNA technology has led to the production of novel varieties and cultivars of tomatoes, i.e., “Flavor saver tomato,” which are much popular and productive for good yield and similar is trend for other crops.

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6.1 INTRODUCTION 6.1.1 PERSPECTIVE OF GENETIC ENGINEERING Horticulture plays an important role in gross domestic product GDP of any country. It is growing fruit crops, flowering plants, vegetables, mushrooms, and medicinal plants (Sonah et al., 2011). Biotechnology has emerged as one of the main fields of biology which includes practices like culturing crops from vegetative tissues, diagnosis of diseases from molecular analysis, genetic engineering, and mutational breeding (Oladosu et al., 2016). By advancement in biology, especially genetic engineering, conventional breeding methods for crops have been altogether changed. Now crops with better characteristics and less costly are available in the market (Thakur et al., 2012). Identification of desirable genes in organisms then isolation of these particular genes of interest and insertion into an organism in which those traits are required is called genetic engineering. Genetic engineering is used to enhance the quality of organisms by transgenic methods (Agarwal et al., 2016). Genetic engineering is more important and precise method for improvement of crop traits than conventional breeding. A wide range of genetic manipulations can be achieved through genetic engineering (Khatodia et al., 2016). Edition of few genes leads to desirable mutations in crops which were previously gained through several experiments and time taking breeding methods (Kim & Kim, 2014). 6.1.2 CONVENTIONAL BREEDING METHODS Traditional crop breeding is advantageous in the sense that it increases the accessibility of genetic makeup, and it may be used for introduction of new characteristics for the improvement of crops. Conventional breeding however is not favorable as genetic diversity (GD) may be lost due to harsh environment and plants may become stressed (Bortesi & Fischer, 2015). Keeping in view the danger of genetic resources depletion, traditional breeding methods is unable to sort out global food shortage. Production of a hybrid plant with desirable traits through combination of several phenotypic characters is somewhat useful to increase crop production and it has been widely used in past but nowadays new breeding technologies are far more efficient, less time consuming and better yielding (Kisaka & Kida, 2019). Recently traditional breeding of plants has been improved by wide

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crosses, wild traits introduction in relative crops, protoplast fusion, double haploid techniques and tissue culture techniques (Cheng et al., 2009). For crop improvement their wild relatives are crossed to achieve better characteristics like higher resistance against biotic and abiotic factors (Harvey, 2009). By identifying crops with better phenotypes and then combining these traits through cross breeding gives rise to new crop varieties with better traits and can institute a new crop lineage with familiar and desired traits. It may sometimes have deleterious effects as new crop lineage may be damaging to local crop varieties (Chen et al., 2018). A particular trait can be enhanced by simple cross breeding with wild plants with superior traits (Diaz et al., 2007). Any desired trait can be introduced into any crop by back cross between selected progeny and plants with superior traits. Back-crossing should be repeated for several generations so as to minimize unwanted phenotypic traits (Faize et al., 2004). Although traditional breeding can be useful for instance, but longterm traditional breeding may result in loss of genetic variability and crops with lesser impact. It will be difficult for scientists to improve production for ever-increasing population if it happens. So, there is a great need to revolutionize crop breeding from traditional breeding to modern breeding techniques involving mutational breeding, speed breeding techniques and rapid generations advances (RGA) (Samineni, 2019). So, it is noteworthy that traditional breeding has some positive effects in crop improvement, but it is greatly realized that modern breeding techniques must be adopted to feed a growing population with depleting agricultural resources. 6.2 CROP BREEDING TECHNIQUES Crop breeding is needed for the improving production of less vulnerable species with high yield and desired characteristics. For this purpose, there are following techniques which are in practice to explore complete gene pool and enhance favorable traits of vegetatively propagated crops. 6.2.1 CLONAL BREEDING (CLONAL SELECTION) Clones are plants produced from a single parent plant without any sexual contact. All the clones are genotypically exactly the same, but due to

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environmental effects, some clones show better growth than others. Plants with better growth are selected and breaded again and again for crop improvement, e.g., potato (De Haan, 2009). 6.2.2 HYBRIDIZATION AND SELECTION Different traits from different but compatible species can be combined in one species with all desirable characteristics. In horticulture, this technique is being widely used to get vast variations in ornamental plants. In food crops, it is also being used for example potato crop cultivars are produced by combining traits from different species (Ross, 1986). Strawberry crop cultivars are also produced with improved yield, texture, and color. Disease resistance genes are also introduced into new cultivars which have enhanced the crop adaptability (Capocasa et al., 2008). 6.2.3 POLYPLOIDY BREEDING Polyploids are plants with heterozygous nature containing the genetic materials from two or more than two relative species. Polyploidy breeding is essential for creating hybrid vigor which have superior traits and yield better than their diploid counterparts. Polyploid sugarcane has more sugar content and large leaves as compared to its diploid counterpart (Lalitha & Premachandaran, 2007). 6.2.4 MUTATION BREEDING Introgression of desirable mutations in plants for improvement in yield and adaptability is called mutational breeding (Bourgis et al., 2008). Plant breeding can be achieved through mutational breeding in four steps. The first step is the induction of mutations in crops through mutagenic agents. Then selection of desired traits and their study is done in mutants. Then M1 to M3 generations are carefully handled to select mutant with the best characteristics. Finally, mutations are identified, and further evaluation is done, and reports are documented or published (Pathirana et al., 2009).

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6.2.5 TISSUE CULTURE TECHNIQUE In vegetatively propagating crops sometimes pathogenic attacks or environmental factors may lead to deleterious effects on desired traits. To avoid such circumstances, propagules are prepared by tissue culture techniques, and disease-free stock plants are incubated for growing. For culturing mostly meristematic tissues are used for stock preparation. This technique is the most effective and rapid technique for vegetative propagation of tomato, potato, and sweet pea (Lebot, 2009). 6.2.6 PHOTOAUTOTROPHIC MICROPROPAGATION It is a method of propagation that involves tissue culture techniques. It is found that chlorophyll containing parts of explants can photosynthesis more effectively if excess of CO2 effective photon flux and required humidity is provided. If all these requirements are met, then there is no need of exogenous nutrient supply to young plantlets. Such plantlets grown in controlled environment in vitro are cost effective, and plantlets are less prone to diseases or stresses. When grown in soil, these plants will grow efficiently and yield much better. This is a widely used technique for vegetative propagation of crop plants including wheat, maize, rice, and cotton (Progress in Biotechnology, 2001; Fischhoff et al., 1987). 6.2.7 INTROGRESSION OF GENES (TRANSGENIC ENGINEERING) Genes with better characteristics are found in wild plants and then introduced into cultivated crops through marker assisted genetic programs. For example, resistance genes in wild species Solanum demissum are introgressed in cultivated species of potato. These potato plants with external genes from wild plants when grown have better tubers appearances and more yield (Lambert & Pinto, 2002). Maize variety has been improved through transgenic breeding method. This maize has higher values of vitamins as compared to others. Vitamin A, C, and E quantities have been increased through transgenic breeding (Newell, 2008). Transgenic breeding has improved the dried biomass of tomato (Dunwell, 2000). Carbohydrate metabolism has been improved through transgenic breeding in soybean (Krimisky, 2019).

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6.2.8 PLANT BREEDS THROUGH MOLECULAR TECHNIQUES Firstly, it is more important to recognize the problem causing gene accountable for causing disorder in living being/plants. To identify faulty genes, molecular markers, ligases, restriction enzymes (REs) and endonucleases are used. Some techniques require modification during transcription of post translational modification to identify the genes manipulating the disorder (Huang, 2016). To develop new crop cultivars with better traits and higher yield, biomolecular markers are widely being used (Schaart et al., 2016). More than 40 million hectares are in use for the cultivation of transgenic crops worldwide. After the production of some useful cash crop cultivars, plant breeding methodology has been revolutionized, and ,nowadays molecular markers are widely used in combination with traditional techniques of breeding. This combination of modern techniques with traditional ones has addressed several issues related to phenotypic and special traits related to disease resistance and higher yields (Vilanova et al., 2012). Molecular markers are useful to identify the sterile male from a given population using molecular approaches. Using molecular approaches, researchers have also identified some genes which have the ability to restore the fertility in cash crops, including rice, sorghum, and maize (Dwivedi et al., 2008). Modern mutagenesis is important to be followed as tradition breeding methods have some limitations like producing undesirable mutation in genome, large population for scaling is required and it is very time and labor consuming (McCallum et al., 2000). 6.2.9 SPEED BREEDING (ACCELERATED PLANT BREEDING TOOLS) Some of the plant species are hard enough to be used in research or breeding programs such species need special accelerated methods of breeding tools which are called speed breeding. During the 1980s a research work carried out by NASA was first time very astonishing till then, researchers are trying these tools for accelerating the plant breeding programs. A research team at Queensland University gives the name speed breeding in the year 2003. These protocols are in practice for several crop species for betterment of the crop genomics (Watson et al., 2018; Ghosh et al., 2018). Speed breeding is a superior technique as it is less time consuming and precise tool for breeding. It is suitable for a wide range of germplasm. It does not need any in vitro methods of culturing as in double haploid protocol it is

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mandatory to culture embryos in vitro for producing exactly homozygous progeny (Samineni et al., 2019). Speed breeding follows the principles of optimum temperature, light intensity, and night and day duration. Ideal conditions for speed breeding are 22 hours light, 22°C temperature of daytime and 17°C temperature for nighttime with high light intensity. All these conditions are optimum for higher rates of photosynthesis, higher rates of photosynthesis in turn promotes early and excessive flowering, which ultimately leads to higher yields in lesser times (Giliberto et al., 2005). Different light patterns can be used to induce flowering in plants. Researchers have developed a positive correlation between red and far-red light intensity and flowering patterns in many crop species including chickpea, lupins, and faba beans. All these species were subjected to different parts of light patterns, and it was found that blue and far-red light patterns promoted flowering in the early stage of development. LED lights with elevated far-red light were seemed to be most effective in promoting flowering (Ribalta et al., 2017). 6.3 GENETIC ENGINEERING IN HORTICULTURAL CROPS Flavor saver tomato was the first transgenic plant which was introduced in the market by Calgene in the year 1994. This genetically modified (GM) tomato was used for saving the freshness and flavor for a longer time after harvest. These tomatoes were rich in glycoside hydrolase enzymes which delay the protein digestion in the cell wall hence walls remain fresh for a longer time after harvest (Slama-Ayad et al., 2019). Pectin methyl esterase gene was introduced in tomato for better color and texture. Genetically engineered tomato plants were produced which can synthesize a special type of flavonoid (Schijlen et al., 2006). Nutritive value of potato can be improved by promoting expression of phytoene synthase gene which is responsible for production of carotenoid and lutein (Chakraborty, 2010). After harvest life of melon can be increased by improving expression of sensory genes (Lin et al., 2004). 6.3.1 GENETIC ENGINEERING FOR VIRAL RESISTANT PLANTS It is possible to introduce plants which have resistance against certain viral strains. It can be done by introducing viral genes for coat proteins or genes

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responsible for prevention of replication. Introducing the replicase gene was helpful to create virus resistant papaya which was able to resist attack of Papaya ringspot (Verma et al., 2016). Other vegetable which was made resistant to specific viruses include Chinese cabbage which was resilient to Turnip mosaic virus. Watermelon was made resilient to Zucchini yellow mosaic virus (Wu et al., 2009; Ghosh et al., 2006). 6.3.2 GENETIC ENGINEERING FOR BACTERIAL RESISTANCE Lytic peptides, glycoproteins for iron sequencing and certain lysozymes are used as antibacterial agents for the production of crops which can tolerate the attack of bacteria (Tarafdar et al., 2014). Antibacterial peptides are especially of interest when thinking about transgenic plants with antibacterial resistance. Modified tomato showed significant resistance against bacterial strains responsible for wilts and spots. This transgenic tomato was prepared by introducing Arabidopsis NPRI gene and it was helpful to counter several kinds of bacterial strains (Lin et al., 2014). Citrus species have been made bacterial resistant by introducing different genes for different resistance. GM citrus prepared by introducing hrpN gene. This gene was responsible for resistance against hypersensitivity, and it also helped for systemic acquired resistance (SAR) against bacteria (Barbosa-Mendes et al., 2009). Antimicrobial proteins including attacin A are helpful against wider class of bacterial as well as attacin A can be produced in large quantity by introducing attacin A gene (Cardoso et al., 2010). AtNPRI gene from Arabidopsis is introduced in plants to regulate SAR in a better way (Zhang & Voytas, 2011). Canker disease causing bacteria Xanthomonas axonopodis cause a lot of damage to the citrus fruits. Cecropin B gene is inserted for resistance against this bacterium. Cecropin B gene is mainly accountable for the generation of bivalent antimicrobial protein which is bacteriostatic and does not allow the bacteria to grow and replicate after incubation to the fruits (He et al., 2011). 6.3.3 GENETIC ENGINEERING FOR INSECT RESISTANCE Insects are responsible for a great damage before and after harvest. Nowadays there are several GM crops which are highly resistant to insect attacks. These modified crops that are incorporated with a gene derived

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from Bacterium thuringiensis (Bt). It is expressed into a crystalline protein which is toxic in nature and hence called Bt toxin (Tsaftaris et al., 2000). Cry genes derived from Bt have been incorporated into eggplant, maize, cabbage, cauliflower, garlic, broccoli, tomato, Chinese cabbage choysum and potato for inducing insect resistance (Jin et al., 2000; Zheng et al., 2004; Cao et al., 2001). These genes are useful to resist a wide range of insect attacks including Leptinotarsa decemlineata, Bemisia tabaci, Macrosiphum euphorbiae and Helicoverpa armigera. Cowpea trypsin is also useful for inducing insect resistance in cauliflower and Chinese cabbage (Zhao et al., 2006). 6.3.4 GENETIC ENGINEERING FOR ABIOTIC STRESS TOLERANCE Drought, salinity, water logging, low, and high temperature, heavy metals, and severe winds may be fatal for crops hence called abiotic stress. Abiotic stress leads to increased antioxidant activity and alters the biochemical makeup of cytosol (Rai & Shekhawat, 2014). For the successful engineering of crops for a biotic stress, it is important to understand the cell-to-cell communication and cell signaling pathways. After the better understanding of signaling pathways, the genes required for such pathways are isolated and inserted into the plants which are desired to be modified (Shepard et al., 2019). Another important method of inducing resistance to abiotic stresses is to identify the products of stresses. Then specific enzymes which can digest these substances are identified and genes for encoding these enzymes are inserted into newer plants for induction of abiotic stress resistance (Bhatnagar-Mathur et al., 2008). Abiotic stress tolerance can be enhanced by expressing transcription factors. Over expression of some transcription factors is found useful for improving abiotic stress tolerance as it has been achieved in banana. GM banana was made tolerant to abiotic stresses by expressing foreign gene Musa WRKY71 derived from Musa spp. (Shekhawat et al., 2011). Grape vines were made tolerant to cold stresses by over expressing (DREB)1B derived from Arabidopsis thaliana (Jin et al., 2009). Osmybb4 gene obtained from rice was introduced in apple for induction of cold and drought tolerance. This gene encoded for a special transcription factor and showed high levels of abiotic stress resistance (Pasquali et al., 2008). Arabidopsis thaliana derived gene AVPI which encodes for H+

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pyrophosphatase showed better growth of root system and ultimately these transgenic tomatoes were able to tolerate water shortage in much better way (Park et al., 2005). Gene for glycine betaine biosynthesis (BetA) was introduced into cabbage which showed salt tolerance (Bhattacharya et al., 2004). 6.4 GENOME EDITING TOOLS Over the years the genome editing tools have been gaining importance in dimensions of research in plant breeding. Nucleic acid editing tools involve engineered nucleases and genome editing technologies which edit the nucleic acid according to breeder or researcher’s choice (Weeks et al., 2016). Targeted genome editing in the plant research has explored many areas of interest, and it comprises the following four steps. • Formulation of an engineered nuclease; • Directing the formulated nuclease construct towards the target site; • Expression of the directed nuclease; and • Analyzation of plants genome to produce desired changes (Curtin et al., 2012). The engineered nucleases constructs can be targeted to achieve various modifications in the genome and all of these genome editing tools work to take advantage of innate DNA repair mechanism. The genome editing tools result in breakdown of duplex in the target DNA and these breaks are repaired by the DNA repair mechanism and in the process the genome is edited. For instance, the nucleases may induce the targeted genome modification and subsequent repair through a mechanism known as nonhomologous end joining (NHEJ). Normally if the double-strand breaks created by the genome editing tools are clean, then the chances of mutation are very low using the endogenous DNA repair mechanism the genome editing is accomplished. However, on certain occasions the frameshift mutation can happen (Maresca et al., 2013). In the coming line, the genome editing technologies are being discussed briefly, which include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered randomly interspaced short palindromic repeats (CRISPR).

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6.4.1 ZINC-FINGER NUCLEASES (ZFNS) Zinc-finger nucleases (ZFNs) are the combination of Fok1 endonucleases and zinc-finger protein. The targeted genome site is efficiently recognized by the ZFNs (Kim & Kim, 2014). The role of ZFNs in genome editing was first reported in A. thaliana and Nicotiana tobacum in 2005 (Wright et al., 2005). The innate DNA repair mechanism is utilized in the genome editing procedures by the genome editing tools like ZFNs. For this genome editing tool to work the nuclease has to dimerize first. ZNFs induce double strand breaks to paralyze dominant mutations. the non-homologous end joining (NHEJ) then repairs the double strands breaks caused by the ZFNs. If the double strand break cut is clean then NHEJ induces no mutation (Osakabe et al., 2010). Sometimes multiple doses of ZFNs can be delivered to completely abort any specific genome part. In Zea mays, the editing of the IPK1 promoter with the help of ZFNs has resulted in herbicide tolerance (Shukaal et al., 2009). 6.4.2 TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES (TALENS) Transcription activator-like effector nucleases (TALENs) belong to REs family that can be customized to edit a specific sequence of DNA. The TALEN tool is a combination of Fok-1 Restriction endonuclease found in some bacteria and Transcription Activator like Effector DNA binding site. The transcription activators like effectors can be custom designed for their targeted manipulation to cut any sort of DNA sequence. The genome editing endonucleases in the form of TALENs edit the genome by activating DNA repair mechanism (Kim & Kim, 2014). The TALENs have been used with great success to modify the genome of horticultural food crops like Potato and Tomato by various researchers working across the globe (Lor et al., 2014; Sawai et al., 2014). TALENs have been used on various occasions to address the genetic maladies and the issue like that. In vitro use of TALENs to manage sickle cell hemoglobin disease has also been reported. However, in perspective of cost and its application expertise requirement puts the TALENs as complicated genome editing tool as compared to others.

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6.4.3 CLUSTERED RANDOMLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR) Clustered randomly interspaced short palindromic repeats (CRISPR) are the loci which contains abundant short palindromic repeats. The CRISPR provides the immunogenic potential against the pathogenic nucleic acid by its targeted degradation by the Cas9 protein. The Cas9 acts as RNA directed DNA endonuclease that degrades the DNA as the target becomes specified (Gaj et al., 2013). The CRISPR genome editing requires the Cas9 component and customized construct of RNA (Jinke et al., 2012). The customized construct of RNA or single guide RNA has a director sequence 19–22 base pairs. This director sequence is often referred as protospacer. The protospacer mutates targets the sequence (Khatodia et al., 2016). The type two system or CRISPR cas9 systems are proving to be efficient genome editing tool. For instance, the CRISPR Cas9 tool is actively utilized as rice plants engineer that changes the tiller angles by editing the LAZY 1 gene (Miao et al., 2013). Similarly, Brooks et al. (2014) reported the genome modification of the tomato plant. The CRISPR Cas9 tool has been used in Citrus sinensis and the targeted gene was CsPDS gene (Jia & Wang, 2014). The transient expression of CRISPR Cas9 in tomato roots has been reported by Ron et al. (2014). The targeted mutation and genome editing has been successfully practiced in a variety of food crops. The work related to manipulation of CRISPR Cas9 genome editing is a topic of researchers worldwide now a days and the tools is proving worth in many plant systems (Khatodia et al., 2016). The technology has opened the way to explore the benefits of genome editing. 6.5 FUTURE ROADMAP OF GENOME EDITING TECHNOLOGY The horticulture practices involving the cultivation of fruits, vegetables, and medicinal plants for the economic benefit have contributed to the farmers and field workers in raising their living standards and economy. To cope with the food demand of current scenarios in perspective of increasing population the horticulture aims to produce the better qualitative and quantitative traits in the plants. The novel genome editing tools are helping the horticulturists worldwide in producing the better yields and better traits in plants. The plants are being GM to increase their nutritional values, enhanced production of secondary metabolites, etc. In the near future the

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plants are expected to be engineered with better traits, and for this purpose, the researcher is expecting more intervention of biotechnology in human life. Genome modification by the help of synthetic genes is not far away, and one cannot deny the fact that synthetic or customized genes will be introduced in the plants to improve their traits (Silva Dias & Ortiz, 2014). Current unwise resource utilization is directing towards food insecurity and resource depletion. Moreover, the future climate change is going to curse the agricultural sector and is going to degrade the farmer even more. For the sustainable agriculture the genome editing tools will be required as they can manipulate the desired genetic change helping to achieve eugenic aims. The sedentary lifestyle of the plants forces them to encounter changes in their immediate vicinity which may be in the form of abiotic stresses and biotic stresses. The future climates are expected to be even harsher, and the plants are expected to encounter these challenges on a more common basis. The genome editing in this regard is also helpful. Plants can be engineered to produce various pharmacological applications and therapeutics (Sharma et al., 2014), like Arabidopsis plants can produce insulin protein in its oilseed (Nykiforuk et al., 2006). Several human therapeutic proteins can be produced using the plants via genome editing tools (Staub et al., 2000). There are successes in manipulating the genome editing tools in a desired manner, and the quality of life is going to improve in the future by the effective utilization of these genome editing tools. Genome editing and production of transgenic organisms has however compromised the safety related issues and their behavior in natural ecosystem is questionable. The transgenic plants producing the oral vaccines is on cards, but the quality and quantity of vaccine in the big picture of human health is yet to be explained (Silva Dias & Ortiz, 2014). Plants produce certain chemicals called phytochemicals or plant secondary metabolites, which are helpful to humans in various aspects. For instance, parsley herbs produce a metabolite apigenin and turmeric plants produce a metabolite called curcumin which has proven to be anticancer on certain occasions (Basey et al., 2015; Wang et al., 2012). The apigenin produced by the parsley is responsible for inducing the apoptosis in human colon cancer cells thereby inhibiting the tumor spread (Turktekin et al., 2011). The curcumin found in turmeric causes the inhibition in cell cycle regulations pathways of malignant cancers (Ravindran et al., 2009). Kim et al. (2008) have reported the anticancerous properties of Cyanidins which is a secondary metabolite actively formed in grapes. The metabolite

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is reported to inhibit the gene expression of COX 2 genes in perspective of human colon cancer cells proliferation. Genomic editing tools can be utilized to produce the GM plants which produce all of these secondary metabolites which act to inhibit the proliferation of the cancer. The abovementioned technologies for the genome edition such as CRISPR/Cas9 may prove fruitful in this regard. Genome editing projects are on the way for several horticultural crops and these projects are going to provide the data about the developmental pathways involved in expression of the genes and thus comparative study of the genome regulation will help further in improving genome editing tools (Sonah et al., 2011). The molecular mechanisms lying behind the expression of genes in the horticultural plants is going to be fruitful in the field of genomics, and this is definitely going to open ways in research related to genomics. The genome editing tools along with the nanotechnology will pave the way for another green revolution which is urgently required against the rising worldwide threats like scenarios of global climate change and food insecurity. 6.6 CONCLUSION The horticulture crops and fruits have promising future due to the advent of DNA engineering technology. The use of DGT has made man to produce fruits and crops with good nutritive and high yield content to cope with the needs of life. Thus, through use of genetic engineering technology (GET) the need of exponential rise of population can be met and the pressure of shortage and low-quality food can be mitigated and eradicated for healthy and safe life. KEYWORDS • • • • • • •

Bacterium thuringiensis clustered randomly interspaced short palindromic repeats effector nucleases nonhomologous end joining systemic acquired resistance transcription activator zinc finger nucleases

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REFERENCES Agarwal, S., Grover, A., & Khurana, S. M. P., (2016). Plant molecular biology tools to develop transgenics. In: Khan, M. S., Khan, I. A., & Barh, D., (eds.), Applied Molecular Biotechnology (pp. 34–60). CRC Press Taylor & Francis Group, USA. Barbosa-Mendes, J. M., De Filho, F. A. A. M., Filho, A. B., Harakava, R., Beer, S. V., & Mendes, B. M. J., (2009). Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker. Sci. Hortic., 122, 109–115. Basey, A. C., Fant, J. B., & Kramer, A. T., (2015). Producing native plant materials for restoration: 10 rules to collect and maintain genetic diversity. Nativ. Plants J., 16, 37–53. Bhatnagar-Mathur, P., Vadez, V., & Sharma, K. K., (2008). Transgenic approaches for abiotic stress tolerance in plants: Retrospect and prospects. Plant Cell Rep., 27, 411–424. Bhattacharya, R. C., Maheswari, M., Dineshkumar, V., Kirti, P. B., Bhat, S. R., & Chopra, V. L., (2004). Transformation of Brassica oleracea var. capitata with bacterial betA gene enhances tolerance to salt stress. Sci. Hortic., 100(1, 4):215–227. Bhattacharya, R. C., Viswakarma, N., Bhat, S. R., Kirti, P. B., & Chopra, V. L., (2002). Development of insect-resistant transgenic cabbage plants expressing a synthetic cryIA(b) gene from Bacillus thuringiensis. Curr. Sci., 83, 146–150. Bortesi, L., & Fischer, R., (2015). The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol. Adv. Bourgis, F., Guyot, R., Gherbi, H., Tailliez, E., Amabile, I., & Salse, J., (2008). Characterization of the major fragrance gene from an aromatic japonica rice and analysis of its diversity in Asian cultivated rice. Theor. and Appl. Genet., 117, 353–368. Cao, J., Shelton, A. M., & Earle, E. D., (2001). Gene expression and insect resistance in transgenic broccoli containing a Bacillus thuringiensis cry1Ab gene with the chemically inducible PR-1a promoter. Mol. Breed., 8, 207–216. Capocasa, F., Diamanti, J., Tulipani, S., Battino, M., & Mezzetti, B., (2008). Breeding strawberry (Fragaria X ananassa Duch) to increase fruit nutritional quality. BioFactors, 34, 67–72. Chen, G., Ye, C. M., Haung, J. C., Yu, M., & Li, B. J., (2018). Cloning of papaya ringspot virus (PRSV) replicase gene and generation of PRSV-resistant papayas through the introduction of the PRSV replicase gene. Plant Cell Rep. 20, 272–277. Cheng, L., Zou, Y., Ding, S., Zhang, J., Yu, X., Cao, J., & Lu, G., (2009). Polyamine accumulation in transgenic tomato enhances the tolerance to high temperature stress. J. Integ. Plant Biol., 51, 489–499. De-Haan, S., (2009). Potato Diversity at Height: Multiple Dimension of Farmer-Driven in Situ Conservation in the Andes. PhD thesis, Wageningen University, The Netherlands. Diaz De La, G. R. I., Gregory, J. F., & Hanson, A. D., (2007). Folate biofortification of tomato fruit. Proc. Natl. Acad. Sci., 104, 4218–4222. Dunwell, J. M., (2000). Transgenic approaches to crop improvement. J. Exp. Bot., 51, 487–496. Dwivedi, S., Perotti, E., & Ortiz, R., (2008). Towards molecular breeding of reproductive traits in cereal crops. Plant Biotechnol. J., 6, 529–559.

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Faize, M., Sourice, S., Dupuis, F., Parisi, L., Gautier, M. F., & Chevreau, E., (2004). Expression of wheat puroindoline- b reduces scab susceptibility in transgenic apple (Malus x domestica Borkh.). Plant Sci., 167, 347–354. Fischhoff, D. A., Bowdish, K. S., Perlak, F. J., Marrone, P. G., McCormick, S. M., Niedermeyer, J. G., Dean, D. A., et al., (1987). Insect tolerant tomato plants. Biotechnol., 5, 807–813. Ghosh, S., Watson, A., Gonzalez-Navarro, O. E., Ramirez-Gonzalez, R. H., Yanes, L., Mendoza-Suárez, M., et al., (2018). Speed breeding in growth chambers and glasshouses for crop breeding and model plant research. Nat. Protoc., 13, 2944–2963. Giliberto, L., Perrotta, G., Pallara, P., Weller, J. L., Fraser, P. D., Bramley, P. M., Fiore, A., et al., (2005). Manipulation of the blue light photoreceptor cryptochrome 2 in tomato affects vegetative development, flowering time, and fruit antioxidant content. Plant Physiol., 137, 199–208. Girhepuje, P. V., & Shinde, G. B., (2011). Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to Fusarium oxysporum f. sp. lycopersici. Plant Cell Tiss. Organ Cult., 105, 243–251. Harvey, A., (2008). Natural products in drug discovery. Drug Discov. Today, 13, 894–901. He, Y., Chen, S., Peng, A., Zou, X., Xu, L., Lei, T., Liu, X., & Yao, L., (2011). Production and evaluation of transgenic sweet orange (Citrus sinensis Osbeck) containing bivalent antibacterial peptide genes (shiva A and cecropin B) via a novel Agrobacterium-mediated transformation of mature axillary buds. Sci. Hortic., 128, 99–107. Huang, X., (2016). From genetic mapping to molecular breeding: Genomics have paved the highway. Mol. Plant, 101–119. Jin, R., Liu, Y., Tabashnik, B. E., & Borthakur, D., (2008). Development of transgenic cabbage (Brassica oleracea var. capitata) for insect resistance by Agrobacterium tumefaciens-mediated transformation. In Vitr. Cell. Dev. Biol. Plant, 36, 231–237. Khatodia, S., Bhatotia, K., Passricha, N., Khurana, S. M. P., & Tuteja, N., (2016). The CRISPR/Cas genome-editing tool: Application in improvement of crops. Front. Plant Sci. 7, 506. Kim, H., & Kim, J. S., (2014). A guide to genome engineering with programmable nucleases. Nat. Rev. Genet., 15, 321–334. Kisaka, H., & Kida, T., (2019). Transgenic tomato plant carrying a gene for NADPdependent glutamate dehydrogenase (gdhA) from Aspergillus nidulans. Plant Sci., 164, 35–42. Krimsky, S., (2019). Traditional plant breeding. In: GMOs Decoded. MIT Press: Cambridge, MA, USA. Lalitha, R., & Premachandran, M. N., (2007). Meiotic abnormalities in intergeneric hybrids between Saccharum spontaneum and Erianthus arundinaceus (Gramineae). Cytologia, 72, 337–343. Lambert, E. S., & Pinto, C. A. B. P., (2002). Agronomic performance of potato interspecific hybrids. Crop Breed. and Appl. Biotechnol., 2, 179–188. Lebot, V., (2009). Tropical root and tuber crops: Cassava, sweet potato, yams, aroids. CABI, Cambridge, Crop Prod. Sci. in Horticul. Ser., 17, 413. Lin, W., Lu, C., Wu, J., Cheng, M., Lin, Y., Yang, N., Black, L., et al., (2004). Transgenic tomato plants expressing the Arabidopsis NPR1 gene display enhanced resistance to a spectrum of fungal and bacterial diseases. Transgenic Res., 13, 567–581.

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McCallum, C. M., Comai, L., & Greene, E. A., (2000). Targeted screening for induced mutations. Nature Biotechnol., 18, 455–457. Newell-McGloughlin, M., (2008). Nutritionally improved agricultural crops. Plant Physiol., 147, 939–953. Oladosu, Y., Rafii, M. Y., Abdullah, N., Hussin, G., Ramli, A., Rahim, H. A., Miah, G., & Usman, M., (2016). Principle and application of plant mutagenesis in crop improvement: A review. Biotechnol. Biotechnol. Equip., 30, 1–16. Park, B., Liu, Z., Kanno, A., & Kameya, T., (2005). Increased tolerance to salt- and waterdeficit stress in transgenic lettuce (Lactuca sativa L.) by constitutive expression of LEA. Plant Growth Regul., 45, 165–171. Pasquali, G., Biricolti, S., Locatelli, F., Baldoni, E., & Mattana, M., (2008). Osmyb4 expression improves adaptive responses to drought and cold stress in transgenic apples. Plant Cell Rep., 27, 1677–1686. Pathirana, R., Vitiyala, T., & Gunaratne, N. S., (2009). Use of induced mutations to adopt aromatic rice to low country conditions of Sri Lanka. In: Induced Plant Mutations in the Genomics Era (pp. 388–390). Proceedings of an International Joint FAO/IAEA Symposium. International Atomic Energy Agency, Vienna, Austria. Rai, M. K., & Shekhawat, N. S., (2014). Recent advances in genetic engineering for improvement of fruit crops. Plant retrospect and prospects. Plant Cell Rep., 27, 411–424. Ribalta, F. M., Pazos-Navarro, M., Nelson, K., Edwards, K., Ross, J. J., Bennett, R. G., Munday, C., et al., (2017). Precocious floral initiation and identification of exact timing of embryo physiological maturity facilitate germination of immature seeds to truncate the lifecycle of pea. Plant Growth Regul., 81, 345–353. Ross, H., (1986). Potato breeding – problems and perspectives. Paul Parey, Berlin and Hamburg. Adva. In: Plant Breed. Ser., 13, 132. Samineni, S., Sen, M., Sajja, S. B., & Gaur, P. M., (2019). Rapid generation advance (RGA) in chickpea to produce up to seven generations per year and enable speed breeding. Crop J., 71–77. Schaart, J. G., Van De, W. C. C. M., Lotz, L. A. P., & Smulders, M. J. M., (2016). Opportunities for products of new plant breeding techniques. Trends Pl. Sci. Schijlen, E., Ric De Vos, C. H., Jonker, H., Van, D. B. H., Molthoff, J., Van, T. A., Martens, S., & Bovy, A., (2006). Pathway engineering for healthy phytochemicals leading to the production of novel flavonoids in tomato fruit. Plant Biotechnol. J., 4, 433–444. Shekhawat, U. K. S., Ganapathi, T. R., & Srinivas, L., (2011). Cloning and characterization of a novel stress responsive WRKY transcription factor gene (MusaWRKY71) from Musa spp. cv. Karibale Monthan (ABB group) using transformed banana cells. Mol. Biol. Rep., 38, 4023–4035. Shepard, J. F., Bidney, D., Barsby, T., & Kemble, R., (2019). Fusion of protoplasts. Biotechnol. Biol. Front., 31–39. Silva, D. J., & Ortiz, R., (2014). Advances in transgenic vegetable and fruit breeding. Agric. Sci., 5, 1448–1467. Slama-Ayed, O., Bouhaouel, I., Ayed, S., De Buyser, J., Picard, E., & Amara, H. S., (2019). Efficiency of three haplomethods in durum wheat (Triticum turgidum subsp. durum Desf.): Isolated microspore culture, gynogenesis and wheat_ maize crosses. Czech J. Genet. Plant Breed., 55, 101–109.

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Sonah, H., Deshmukh, R. K., Singh, V. P., Gupta, D. K., Singh, N. K., & Sharma, T. R., (2011). Genomic resources in horticultural crops: Status, utility and challenges. Biotechnol. Adv., 29, 199–209. Tarafdar, A., Kamle, M., Prakash, A., & Padariya, J. C., (2014). Transgenic Plants: Issues and Future Prospects, 2, 1–47. Thakur, A. K., Chauhan, D. K., Parmar, N., & Verma, V., (2012). Role of genetic engineering in horticultural crop improvement: A review. Agric. Rev., 33, 248–255. Tsaftaris, A., Polidoros, A., Karavangeli, M., Nianiou-Obeidat, I., Madesis, P., & Goudoula, C., (2000). Transgenic crops: Recent developments and prospects. In: Balázs, E., Galante, E., Lynch, J. M., Schepers, J. S., Toutant, J., Werner, D., & Werry, P. A. T. J., (eds.), Biological Resource Management Connecting Science and Policy (pp. 187–203). Springer Berlin Heidelberg, Berlin, Heidelberg. Verma, R. K., Mishra, R., & Gaur, R. K., (2016). Potato virus Y genetic variability: A review. In: Gaur, R. K., Petrov, N. M., Patil, B. L., & Stoyanova, M. I., (eds.), Plant Viruses: Evolution and Management (pp. 205–214). Springer, Berlin, Germany. Vilanova, S., Cañizares, J., Pascual, L., Blanca, J. M., Díez, M. J., Prohens, J., & Picó, B., (2012). Application of genomic tools in plant breeding. Curr. Genomics, 13, 179–195. Wang, H., Oo Khor, T., Shu, L., Su, Z., Fuentes, F., Lee, J., & Tony, K. A. N., (2012). Plants vs. cancer: A review on natural phytochemicals in preventing and treating cancers and their drug ability. Anti-Cancer Agents Med. Chem., 12, 1281–1305. Watson, A., Ghosh, S., Williams, M. J., Cuddy, W. S., Simmonds, J., Rey, M. D., Asyraf Md, H. M., et al., (2018). Speed breeding is a powerful tool to accelerate crop research and breeding. Nat. Plants, 4, 23–29. Wu, H., Yu, T., Raja, J. A. J., Wang, H., & Yeh, S. D., (2009). Generation of transgenic oriental melon resistant to zucchini yellow mosaic virus by an improved cotyledoncutting method. Plant Cell Rep., 28, 1053–1064. Zhang, C., Wang, T., Zhang, Y., Wang, A., Zhang, Y., Lin, K., Li, C., et al., (2014). Genomic analyses provide insights into the history of tomato breeding. Nat. Genet., 46, 1220–1226. Zhao, J. L., Xu, H. L., Zhu, Z., & Liang, A. H., (2006). Transformation of modified cowpea trypsin inhibitor gene and antibacterial peptide gene in Brassica pekinensis protoplasts mediated by Agrobacterium tumefaciens. Euphytica, 149, 317–326. Zheng, S. J., Henken, B., Kyun, A. Y., Krens, F., & Kik, C., (2004). The development of a reproducible Agrobacterium tumefaciens transformation system for garlic (Allium sativum L.) and the production of transgenic garlic resistant to beet armyworm (Spodoptera exigua Hübner). Mol. Breed., 14, 293–307.

CHAPTER 7

Genetically Engineered Microorganisms PRAFUL UPENDRA SAHA,1 AHMAD ALI,1 and JOHRA KHAN2,3

Department of Life Sciences, University of Mumbai, Vidyanagari, Santacruz, Mumbai, Maharashtra, India 1

Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Majmaah University, Majmaah, Saudi Arabia

2

Health and Basic Sciences Research Center, Majmaah University, Majmaah, Saudi Arabia

3

ABSTRACT Humans have used microorganisms for many works such as food production, used in environments for a stable nature by enhancing the plants and crops in their areas. These microorganisms need to be altered so that these microorganisms would be susceptible to a particular situation. So, microbiologists and scientists discovered a sure way to alter the enzymes and genes in these Microorganisms. Thus, the term genetically engineered microorganisms (GEMs) come into life. The scientists took over hundreds of years after the discovery of human DNA. They started to improve the use of microorganisms by using several techniques to genetically alter them to make them more efficient in especially food production. The modification has been prepared to the hereditary makeup of the microorganisms to either create a new form of protein or a new food product in food processing to improve or enhance the existing protein/enzyme. Some techniques involve mutagenesis and interspecies hybridization to change

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the genomic makeup of the microorganisms. Many achievements have been made due to this technique in molecular biology and biochemistry over the past few years. 7.1 INTRODUCTION Microorganisms are used for many purposes throughout the human race as a part of their daily life. These microorganisms have been used for many purposes, produced in nature and by artificial techniques known as a xenobiotic compound. These are used in many sectors such as food, agriculture, industrial, medical, environmental, and research sectors. Microorganisms include five primary types: bacteria, fungi, protozoa, archaea, and viruses (Meselson & Radding, 1975). These microorganisms are used for the genetic recombination process to make the changes in these organisms, which brings about changes in these microorganisms and through transfection and transformation process these microorganisms will get the specific gene, which is inserted into them and therefore they derive, for that specific protein (Su et al., 2016). This method that is used for the genetic recombination of the microorganism has been successful in all the human sciences as they are using these technologies to create changes in the system of medicine, in the food sector, in agriculture by infecting or inserting the genes required into the organisms and then inserting into the cells which bring about changes in the particular organism. They exhibit only that protein or properties they are coded (Martin et al., 2017). These methods are used for many gene targeting purposes, therefore used in gene therapy in medicine. The modified microorganisms are mostly called genetically engineered or GM ones as a common term used by the local language. Therefore, these engineered organisms are used in many food productions (Brick et al., 2012). The organisms which are GM are beneficial and deprived of many harmful threats the environment exhibits. Moreover, this makes the organisms disease-free as well as high in nutritional content. These genetically engineered microorganisms (GEMs) are used in food to produce specific enzymes responsible for producing many food substances like cheese, yogurt, etc. Furthermore, the GEMs are helpful in the bioremediation in the environment by degrading certain harmful substances such as plastics, many harmful pollutants which cause toxicity in the environment and also in the soil, which lead to lower life

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quality in plants and thus decrease the overall output of the fruits and vegetables and other substances from them (Charlesworth et al., 2009). This chapter covers the basics of genetic engineering as this method is used for the creation of GEMs and also the microorganism which is being engineered by using these processes. 7.2 MICROBIAL GENETICS

Studying microbial genetics is necessary to understand the microbial pattern, and their relationship with the environment is central for understanding the evolution of microbial genetics. There are primarily five main types of microorganisms used for microbial genetics, so genetic engineering is applied accordingly (Paigen & Petkov, 2018). 7.2.1 BACTERIA Bacterial genetics includes the studies related to the information on their genetic history, mechanisms of their chromosomes, their plasmids, phages, and transposons. The most common study used in bacteria is gene transfer, which includes transforming the genetic material, conjugation, and transduction (Van Os et al., 2006). The transformation process is an adaptation of the bacteria for transferring the DNA within cells using an interceding medium. It is a complicated process, and it requires energy to adapt to the process of DNA damage repairing (Morel et al., 2011). Conjugation is a process in bacteria that includes the transfer of the genetic material which is taken place between two bacterial cells. The conjugation process has been primarily used in the bacteria Escherichia coli (E. coli). It has also been studied in bacteria Mycobacterium smegmatis (Zhang et al., 2010). It is a stable process used by most researchers and scientists for introducing a foreign gene in the genome of the host’s cells (Anderson & Schneider, 2007). 7.2.2 ARCHAEA Archaea comes under the category of unicellular and prokaryotic organisms, i.e., they do not have any cell nucleus or other cell organelles in

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them. They replicate asexually by the process of binary fission. They have a typical relationship with the bacteria, although they are more closely concerning the eukaryotes because they contain tRNA. From the studies, it has been found that some Archaea species can survive in extreme environments, which has led to the idea of many applications of them in genetics like Archaeal enzymes (Anderson & Schneider, 2007). The studies in Archaea include the Gene transfer as well as genetic exchange between different Archaea. Archaeal Genetics includes the study of genes in a cell that does not contain a nucleus. They only have single, circular chromosomes, which contain many origins for the replication process to initiate the synthesis of DNA. Archaea and bacteria are similar in their structures because they either have a spherical shape known as coccus or a rod-like shape known as bacillus. Archaea and eukaryotes have almost identical ribosomes used for protein synthesis (Fusaki et al., 2009). Archaea can live in extreme and harsh environments with acidic pH like in oceans, in humans, in the gut of the ruminant, and in the lake, which contains a high amount of salt; therefore, they are known as extremophiles (Kaji et al., 2009). 7.2.3 FUNGI Fungi are either present as unicellular or as multicellular organisms. Fungi are microbes used to break down the organic matter in the surroundings by secreting an enzyme from them (Anderson & Schneider, 2007; Khan et al., 2015). The genetic study of Fungi uses yeast and filamentous fungi used as model organisms for genetic research in eukaryotic research, including the regulation of cell cycle, regulation of gene, and the study of chromosome structure. The gene functioning was led by the studies of the fungus Neurospora crassa. This fungus is used as a model organism because it is straightforward to grow, and as this fungus has a haploid life cycle, so it shows only the recessive traits in its offspring. Moreover, therefore the genetic analysis becomes simpler in these organisms. N. crassa (Neurospora crasa) is found in the tropical and subtropical regions in its environment. It was also found mainly on the dead plant remains after the fire (Khan et al., 2017). Neurospora fungi were used by scientists Edward Tatum and George Beadle, who use them in their experiments. They won a Nobel Prize for

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their work on these Neurospora fungi. The experiment of these fungi species led to the hypothesis of one gene-one enzyme, which states that specific gene codes only for those specific proteins. It was a significant setback in molecular biology due to which developments have taken place in this field (Alotaibi & Khan, 2016; Strauss, 2016). 7.2.4 PROTOZOA

These are the unicellular organisms that contain a nucleus and tiny cellular bodies in their cytoplasm. These cellular bodies are ultramicroscopic in size. Protozoa have similar flagella to human sperm flagella. Therefore, their studies can help to understand human genetics as well. Paramecium studies have resulted in some understanding of the role of the meiosis process. Paramecia have diploid micronuclei either in 1 or 2 in number and a polyploidy macronucleus (Chan et al., 2005). This macronucleus expresses the genes needed for the cell’s daily functioning, controlling all the non-reproductive cell functions. On the other hand, the micronucleus is a germline nucleus or a generative part that contains the hereditary material that passes from parents to their offspring. In clonal aging, which occurs due to the asexual fission growth phase, in which the cell divisions are occurred by the mitosis process, there is a drastic increase in the DNA damage during successive clonal cell divisions. This process is observed in the protozoan Paramecium tetraurella. When these clonally aged paramecium species are stimulated to undergo the meiosis process, the progeny produced is rejuvenated so that they will undergo many binary fission divisions. In these rejuvenation processes, there is a repair in these DNA damages in the micronucleus of these protozoan species during the process of meiosis (Vanloqueren & Baret, 2017). 7.2.5 VIRUSES Viruses are organisms that are encoded with capsids. These virus organisms are composed of proteins and nucleic acids. They can reassemble in the host cell after the replication takes place in them by themselves. There are several types of viruses that have the ability for genetic recombination. In this, when two or more viruses of the same type infect the same cell, so

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their genomes can recombine to each other to produce another recombinant progeny of those two viruses. They undergo homologous recombination repair to produce a viable progeny, and therefore, this process is known as multiplicity reactivation. So, the enzyme employed in these processes functions homologous to the enzymes employed in bacteria or eukaryotes (Forterre & Prangishvili, 2009). The process of multiplicity reactivation has been found in the pathogenic virus such as influenza virus, adenovirus simian virus, HIV (human immunodeficiency virus), herpes virus, vaccinia virus, and reovirus, as well as in many bacteriophages. 7.3 GENETIC ENGINEERING 7.3.1 HISTORY Genetic Engineering was a term introduced in the late 1970s that was used to describe the rDNA field. As we all knew, recombinant DNA (rDNA) is used by DNA cloning, which is used to grow them in bacteria, and this has evolved from field to field, and the uses of rDNA have been more advanced in the years, so genetic engineering has been taking place. Genetic Engineering uses rDNA technology to alter an organism’s genetic makeup. This process involves manipulating directly one or more genes (Nicholl, 2008). A gene from another species or organisms is mainly added to target organisms or species to give it a phenotypic effect as desired. The term refers to many other techniques used to modify or manipulate organisms or species, including the process of hereditary and reproduction. This term includes artificial selection and other biomedical methods, including in-vitro fertilization, cloning, artificial insemination, and gene manipulation. Because of recombinant technology, the term has become more specific (Schurman & Munro, 2006) (Figure 7.1).

FIGURE 7.1

Production of plasmid DNA.

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The possibility of rDNA technology has come together when finding the restriction enzymes (REs) in 1968 (Berg & Mertz, 2010). The scientist who discovers this restriction enzyme was a Swiss microbiologist, Werner Arber. In the same year, the restriction 2 type enzymes were discovered, leaving the specific site within the DNA, which was essential for genetic engineering techniques. Genetic engineering was based on recombination, done in 1973 by American biochemists Stanley N. Cohen and Herbert W. Boyer; they were among the first to cut the DNA into small fragments and rejoiced different fragments and then attractive in that new set of genes into E. coli bacterial species, which was then replicated (Johnson et al., 2005). 7.3.2 PROCESS AND TECHNIQUES Multiple techniques can be used to accomplish genetic engineering. There are several steps Richard has done to create a genetically modified organism (GMO). The researchers who won to create AJ GMO first must choose words in Davis to insert, modify or delete. Then, the gene must be isolated and corporate along with the other elements such as vector. This vector works as a host which carries the gene in another organism. For animals, the gene which is incorporated with the vector is inserted into the embryonic stem cells. While in plants, the gene can be inserted in any tissue in the plant and can be cultured to develop into a fully developed plant as the plant shows totipotency (Rajasekaran et al., 2020). Some included techniques are the microinjection technique, biolistics which is also known as a gene gun. Advancement has taken in this field due to the development of polymerase chain reaction (PCR) and sequencing techniques and the discovery of REs and DNA ligases (Gitschier, 2009). Other tests are done on those particular organisms in which the gene is inserted to ensure stable integration of the gene, proper inheritance, and expression of that particular gene-phenotype. The resultant offspring which is produced is always heterozygous. Many traditional techniques are used for inserting genes into the host genome. The targeting system includes meganucleases and zinc finger nucleases (ZFNs). Many accurate techniques have been developed since 2009, which are more accessible systems than the previous ones. The new techniques involve TALENs (Transcription activator-like effector nucleases and CRISPR (Clustered

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regularly interspaced short palindromic repeat). This CRISPR technique changes living organisms’ genetic sequence by making particular alterations in their DNA (Gaj et al., 2013). Basic processes which are involved in genetic engineering are described briefly in subsections. 7.3.2.1 CHOOSING TARGET GENES This is the initial step of the Genetic Engineering process. In this process, the target genes are identified to insert into the host organism. Furthermore, once that particular gene is found, then the genetic information is inserted into an organism, mostly a bacterium used for storage and modification of that particular bacterium. This leads to the formation of genetically modified (GM) bacteria. Bacteria are primarily used in this process because they are very cheap and are readily available, they tend to reproduce or multiply at a rapid pace, and they are very easy for the transformation process. Once the modified bacteria are stored at–80°C, they can be used for study an infinite number of times, and research can be done on them. Another technique can be used, such as Genetic screening, which is carried out to check for the best gene possible and the organism suited for that particular gene to insert. It involves DNA mutation with several chemicals or any radiation, and then the selection took place. Forward genetics and reverse genetics are used in this screening process (Gaj et al., 2013). In forward genetics, they look at that desirable phenotype and identify the responsible gene for that phenotype. While in Reverse Genetics, the first target a specific gene that is to be mutated, and then they observe the resultant phenotype (Sun & Zhao, 2013). 7.3.2.2 GENE MANIPULATION The DNA or gene is isolated from the cell or any organism, and then other genetic elements are inserted into that gene to allow that gene to be expressed in the host organism. Gene manipulation involves specific techniques, which include Cell Extraction in which the cell is being opened, which causes the DNA to be exposed, and then the DNA is separated from other cell constituents (Lei et al., 2012). Then nucleic acid is purified using the phenol-chloroform process. And then, the nucleic acid is then precipitated from the solution using ethanol or isopropanol. Moreover,

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the screening process is done from the DNA library prepared from that particular gene to check for the desired phenotype. Techniques such as REs cleaving are used in the gene of DNA sequence is known and then isolated. DNA strand is visualized using ethidium bromide stain and then checked under UV light (Wei et al., 2013). An advanced technique known as PCR is used to isolate genes of which the sequences are known. Modification steps include the mixing of the gene with other genetic elements by which it can function appropriately. Construction of genes using rDNA techniques such as restriction digestion ligates and molecular cloning is followed before intersecting the particular DNA into the host (Lei et al., 2012; Mao et al., 2013). 7.3.2.3 INSERTING DNA INTO HOST Many techniques are used to insert the gene into the host organisms. Organisms such as eukaryotes, the transgene, which is inserted, pass to its progeny. Techniques such as Transformation, Transfection, Transduction, and Regeneration are used to insert the gene in which the gene is inserted in its way. In Transformation, the genetic components are altered, bypassing the genetic material from an outside source. This process is taken through the cell membrane. Mainly the bacterial cells are used in the transformation process. The bacteria are treated with heat shock or electroporation, making the bacteria’s cell membrane capable of taking the DNA from the outside source. Calcium chloride solutions are also used to disrupt the bacteria’s cell membrane partially, and then the rDNA material is inserted in the host cell (Li et al., 2014). Transformation is also taken in plants by the process of biolistics, also known as a gene gun. In these processes, the DNA is coated with gold or tungsten, and then the DNA is shot into the embryos of the plant. Plant cells can also be transformed using the electroporation technique, in which an electric shock treatment is used to create the cell-permeable for the plasmid DNA. Another technique is Transfection, which is used explicitly for inserting foreign DNA in animal cells. Many techniques are used in-vitro (Chen & Gao, 2014). These techniques are primarily used for gene therapy, where chemical techniques are used for inserting the gene into cells. Calcium phosphate is used, which makes the cell exposed to the cultured cell. Liposomes and other polymers are used as a vector to transport the DNA in the cell. Biolistics

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and electroporation techniques are also used for transferring genes in the host cell but are not used chiefly. Transgenic animals are also created by using these processes by using the technique of microinjection. The DNA is transferred to the cell nucleus by injecting the DNA through the cell’s nuclear envelope. Transduction is another process of transferring the DNA into the host, but the vector used is a virus. The virulent gene from the virus is removed, and the outside gene is inserted in them. Viruses like the retrovirus and adenovirus are the most popular viral vectors used (Hoban et al., 2016). 7.3.3 APPLICATIONS Genetic engineering has been used in many areas like medicine, industrial research, and the agricultural field in which testing of crops and making changes in the genetic makeup of the crop to provide a disease-free crop, a crop with high nutritional content. Genetic Engineering is also used in plants, animals as well as microorganisms. Microorganisms such as bacteria have been GM, used as an enzyme coding or gene having medicinal properties in bacteria used to treat diseases. In addition, the genetic modification of bacteria produces enzymes that are used for food processing. Plants have also been modified, which can lead to high tolerance properties, have high nutritional content, have resistance to viruses, herbicide, insecticide, and produce edible vaccines (Vanloqueren & Baret, 2017). Genetic engineering has many applications in the medicinal field, including drug manufacturing and gene therapy to find a solution for certain diseases, creating model animals used and modified to replicate normal human conditions. Many applications are used in medicine, such as the production of human insulin, which has led to many successes and is followed by the essential human hormones such as Growth hormones, follicle-stimulating hormone, antibodies production, production of human vaccines. Genetic engineering has also been used to make transgenic mice, the most common animal used for genetic engineering to study many human diseases such as cancer, heart diseases, comorbidities such as arthritis, obesity, and effects of certain substances on the human body. The use of pigs in genetic engineering has been done and found that the probability of transplanting an organ from pig to human is one of the most

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successful and tremendous changes in human medicine (Rai & Ingle, 2012). Genetic engineering has also been used in a research study which is an essential tool for scientists for creating transgenic animals or organisms, which is mainly used for the study for any gene alteration activity or to investigate the particular gene which causes the mutation. Bacteria are also being modified by adding genes into the bacteria using a different process. Because of the many advantages of bacteria economically and in their life cycle, bacteria are the most used microorganism in the Genetic engineering process (Fernandez-Cornejo et al., 2014). Genetic engineering has a role in the industrial sector in which medicines and food processing components are produced by altering the protein’s makeup in the bacteria. The industrial fermentation process is used to grow the organism in a bioreactor and then purify the protein. In food, examples of chymosin protein for making cheese are included. The industrial sector is also used for the production of fuels. Environmental uses such as Bioremediation and Bemoaning are also seen in the Industrial approach of genetic engineering (Curran, 2012). Many issues have been seen in using these processes in another field. Issues including ethical, ecological, and economic issues have been seen in many sectors. The use of GM crops has led to questioning the safety of the crops produced and other environmental issues that can be faced due to these crops. Even ordinary people say that scientists are playing God by altering the genome of organisms. GM foods have their concerns in which one does not have the assurance that whether the food is safe or not whether it led to any reaction in the body. A false belief that the gene from that particular food will mix with the human gene eating that food (Offringa et al., 1990). Moreover, many more issues have been seen in producing genetically engineered organisms (GEOs) (Wilson & Fuchs, 2008). 7.4 GENETICALLY MODIFIED ORGANISMS (GMOS) 7.4.1 INTRODUCTION Genetically modified organisms (GMO), also known as genetically engineered organisms (GEOs), are organisms whose genes have been altered by using genetic engineering. Many people get confused between GMOs and GEOs (Rajasekaran et al., 2020; van Regenmortel et al., 2000). GEOs

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are termed when biotechnology is used to manipulate the genome of organisms directly. According to the United States Department of Agriculture, which is USDA termed GMOs as plants or animals which the genomic sequence has changed with the help of genetic engineering methods and on the other hand, GEO was termed as an organism in which the genes are introduced, eliminated, or are arranged again using rDNA technology like transgenesis (Wilson & Fuchs, 2008). 7.4.2 PRODUCTION OF GMO It takes many processes to create a complete GMO. Initially, the genes are first isolated, which is to be inserted into the target or host organisms. So, the gene can be from any cell source or can be synthesized artificially. And then, the gene is combined with other elements like adding promoter and terminator regions and selectable markers. After that, numerous techniques are used to insert the gene into the host organism or genome. For example, bacteria are induced to take foreign DNA when exposed to heat shock or electroporation techniques. In animal cells, the microinjection technique is used in which the gene is inserted directly into the cell nuclear envelope into the nucleus of the cell. Alternatively, a viral vector is used so that they cause mutation. In plants, the DNA is inserted using the Agrobacterium-mediated recombination technique or gene gun technique, or electroporation technique is used. Since only a single cell is being infected to be transformed using the genetic material, it is a must for that specific cell to generate into a whole organism (Lee et al., 1990). In animals, for ensuring the regeneration phenomena, the DNA is inserted in the embryonic stem cells. Moreover, in plants, the whole organism is produced using the tissue culture technique. Then, tests like DNA sequencing and PCR confirm that the cell contains the new gene set (McFarlane, 2008). To target the exact location of the DNA in the genome, Gene targeting techniques are used in a double-stranded break. Then, Gene editing is used to create the breaks at the specific points, which uses an artificial nuclease engineered in laboratories. The engineered nucleases are of four families: Transcription activator-like effector nucleases (TALEN), the Cas9 guide RNA system adapted from the CRISPR technique, the meganucleases, and the ZFNs. In these four classes, the TALEN and CRISPR techniques are most commonly used.

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The TALEN technique has an excellent specificity for the target, while the CRISPR is very efficient to use and is very easy to design (Anand et al., 2019). Many organisms have been modified using this technique. Most of them are likely to be the bacterium, a microorganism, and bacteria modification is more straightforward to process than another organism because of its cost and high multiplication rate. Even plants and animals have been genetically modified (GM). There are many uses of GMOs in science, agriculture, research, and medical fields. Some of the applications of the GMO in various fields have been mentioned as they have become a part of everyday life, GMO had entered society through the agricultural sector, environmental management system, in medicine as well used in the research sector, which is mentioned. 7.4.3 GMOS IN AGRICULTURE SECTOR The first approved GM foods for human consumption was approved in 1994 by the United States, and so as the progress goes on in GM foods, by 2014, many other food crops such as corn, cotton, and soybeans were GM. About 90% of these crops were planted by 2014–2015 (FernandezCornejo et al., 2014). Genetically engineered crops, as found by studies, reduce the use of chemical insecticides. For example, an insecticide decline in many crops of potatoes, cotton, and corn has given a gene from the bacteria Bacillus thuringiensis (Bt), and this bacterium produces a natural insecticide known as Bt toxin. So, the crops engineered with Bt toxin have a high yield compared to non- Bt engineered crops such as Bt-cotton. Pests such as cotton bollworm (Helicoverpa armigera) and pink bollworm (PBW) (Pectinophora gossypiella), which are the major cotton pests, have also resisted due to the Bt-crops, and therefore the yield was higher (Pray & Huang, 2003). Herbicide-resistant crops or HRC have also been produced, which have the resistance to chemical herbicide specific to particular crops example, in weeds crops, HRC crops treated with specific herbicide can survive in the field. Another example of GM crop is the golden rice explicitly produced for the Asian population, and the crop contains 20 more beta-carotene than the non-GMO one. The golden rice was produced by modifying the original rice with the gene from daffodil Narcissus pseudo narcissus, which produces the enzyme

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known as phytoene synthase enzyme, as well as inserting a gene from the bacteria Erwinia uredovora. These bacteria produce phytoene desaturase enzymes. When inserted into the rice, this gene turns on the beta carotene in these crops, which, when taken in the body, turns to Vitamin A in the liver of humans. Like these crops, many other crops around the globe have been tested with another gene to modify them n make them capable of withstanding extreme weather conditions expected in some areas (Prado et al., 2014). 7.4.4 GMOS IN MEDICINE AND RESEARCH SECTOR GMOs in these years have become one of the significant fields in biomedical studies and research, and it has been followed since the 1980s. GM animals have been made as models to study human genetic diseases, test the therapies, and check for the possible candidates and the risk factors of the particular diseases. Microbes, plants, and animals that are genetically engineered have led to an increase in the sector of pharmaceutical production. They allow the generation of safe and cheaper vaccines and other therapeutics in the medical sector. For example, in Pharmaceutics, the recombinant hepatitis B vaccine was developed by the GM baker yeast and the development of injectable insulin in people with diabetes produced by GM E. coli, a bacterium (Karimi et al., 2002). Moreover, many other vaccines or therapeutics used for conditions such as heart attack or stroke in patients in hemophilia are being produced in the laboratory using GM mammalian cells. In addition, genetically engineered plants are also used for edible vaccines that are under production. These edible vaccines are a protein produced in the plant’s parts that are consumed, so these vaccines, when absorbed in the bloodstream and when these parts are eaten, get absorbed in the body (Thomson, 2001). Thus, it stimulates the immune system for antibody production against specific antigens. These vaccines thus enable a safer way that is painless and are cost-effective in the countries which are less developed in the medicines sector (Tharmalingam et al., 2015). Insects that have been genetically engineered have been a significant step in the research sector. These GM insects have been used to prevent parasitic diseases. Examples of this GM insect are the GM mosquitoes that have been developed. Potato, which is developed, expresses a protein

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called SM1. These proteins block the entry of the malaria parasite, which is plasmodium, into the mosquito’s gut (Wong et al., 1985). This results in the disruption of the life cycle of that particular parasite and renders the mosquito’s malaria resistant. So, when these mosquitos are introduced in the wild would help to reduce malaria transmission in that particular area. Also, genetic modification in human beings is carried via gene therapy technique which has become a treatment option for the spell for many diseases. So, in gene therapy, stem cell technology is coupled with the rDNA method. These allow the stem cell derived from the patient to modify in the laboratory and then be introduced in the desired gene. For example, in conditions like sickle cell anemia, a normal beta-globin gene is introduced in the DNA of bone marrow, a Derived hematopoietic stem cell from the patient who has sickle cell anemia. So, when these GM cells are introduced into the patient, they can cure the disease without any need of a donor (Sugawara et al., 2015). 7.4.5 GMOS IN ENVIRONMENTAL MANAGEMENT A GMO is also applied for the management of different environmental issues. For example, some bacteria are used to produce biodegradable plastics, so they transfer that particular bacteria’s ability to the microbes that are then grown in the laboratory. So, they enable a wide-scale greening of the plastic Industry. In the early 1990s, a British company develops the microbially produced biodegradable plastic call Biopol (polyhydroxyalkanoates or PHA). This plastic was made using a GM bacterium, Ralstonia eutropha, which converts glucose and a variety of organic acids into a flexible polymer (Saxena et al., 2020). GMOs also provide an efficient bioremediation strategy when endowed with the bacterially encoded ability to metabolize oil and heavy metals (Teuscher et al., 2006). 7.4.6 CONTROVERSY IN GMOS PRODUCTION Even though GMOs have many benefits globally, many controversies have been made about GMOs’ risks. This is primarily highlighted in the food sector. Many critics are there of GM crop’s dangers and health risks associated with them. Like allergic reactions, they can cause genetic manipulation in these crops. As studied, GM rice, which is golden rice,

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provides many health benefits to humans by providing them an adequate amount of nutrients, which increases overall health, but it is still unclear. The main risk was the genetically engineered crops gene to the flora in their native place, which can lead to the Evolution of the superbugs, which are resistant to insecticides (Van der Sluijs, 2007). Many cases against GMO production in the food sector in the 1990s, which the European Union (Wong et al.) has addressed. There have been laws made for GMO foods that all the GMO foods should be labeled, which have a proportion of GM products more than 0.9%. However, even after these cases, the consumer and other health groups, also the scientific panels such as Food and Drug Administration (FDA) from the United States, concluded that GM foods are safe and can be consumed without any problems. Even in the Medicines sector, there has been a debate about using GMOs. Like the genetic researchers believe that they are producing GMOs and using gene therapy to cure diseases and ease the suffering of individual suffering to that particular disease, people also thinks that these gene therapy applications will one be used to produce designer babies or to increase the life span of human which can be against natural law. Like every other technology, gene therapy and GMOs are used efficiently in the field and address all the problems, but they should all those techniques very wisely (Satterfield et al., 2013). 7.5 GENETICALLY ENGINEERED MICROORGANISMS (GEMS): OVERVIEW 7.5.1 INTRODUCTION A microorganism known as Microbes has been used throughout human history for many purposes like the production of food substances, medicinal use, and environmental fields, Like in the process of Bioremediation. The most common microorganisms used for the genetic engineering were the bacteria since it has many advantages like they are very cheap, they are straightforward to grow, multiply quickly, are relatively easy to transform, and have clonal properties. Moreover, they can be stored at minus 80°C temperature all the time. So once a gene is isolated, it can be stored in the bacteria and provide a never-ending supply in the research field. So, bacteria are primarily used because it contains a plasmid DNA in which changes can be made according to the use. The other genetic material

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information from other organisms is added to the plasmid, and then they are inserted into the bacteria. Then these bacteria are further stored and modified. Many custom plasmids are used to make the manipulative DNA, and they are cut from the bacteria very quickly. Because of their easy availability, Bacteria have become a great tool these days for scientists who can study a functioning gene and study the gene evolution. Many DNA manipulations take place in the plasmid of the gene and then transfer them to another host (Wu et al., 2008). The use of GEMs is primarily seen in the environmental technology sector (Noori & Chen, 2003). According to the studies, there are different types of contaminants in the surrounding; for example, the plastics used are harmful to the environment, so biodegradation of those plastic substances is significant as in nature, it takes much more time to degrade completely. Genetically engineered microorganisms (GEMs) are made in the laboratory by changing the genetic material, which is plasmid in this sense. So, the genetic materials are introduced in such a way that they are capable of biodegrading harmful substances. The processes through which GEMs are created are taken through rDNA techniques. This rDNA technology uses the plasmid in the bacteria, which is the extra chromosome DNA. Plasmids can replicate independently. Therefore, for the degradation of a particular compound, we need a specific plasmid. For example, plasmid OCT is used to degrade Octane compounds, XYL plasmid for Xylene compounds degradation, CAM and NAH for Camphor, and Naphthalene compounds degradation. Therefore, the use of plasmid is particular (Chaparro‐Riggers et al., 2007). 7.5.2 HISTORY A microorganism was discovered in the period of 1665 to 1885 by Robert Hooke and Antoni Van Leeuwenhoek. Their discoveries include the depictions, observations as well as descriptions of the Microorganisms. The major setback for GEMs came in 1981 when the bacteria were discovered, breaking down certain compounds such as CAM and Salicylate. The second significant breakthrough was found due to the identification of the ‘Superbugs.’ These Superbugs can transfer their plasmids to other genes, and therefore, they were helpful in the breakdown of specific contaminants.

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7.5.3 CRITERIA AND APPROACHES FOR MAKING GENETICALLY ENGINEERED MICROORGANISMS (GEMS) GEMs are usually designed based on their interaction between antibiotic compounds and the contaminant present in the surrounding we want to break. The xenobiotic compounds are manufactured compounds. So, the way the compounds react with the contaminants gives the idea of how the GEMs can be produced and which changes should make in them— also, depending on their Genetic Bases, which include their genetic material interactions. We also have to see the biochemical mechanisms of those Microorganisms or the other compounds to see their ability to break down or degrade those particular compounds. Finally, we have to check for the operon structure of the particular bacteria we want to engineer This is a crucial step to check for any damage in the operon since it can lead to irregularity in protein synthesis (Castéra et al., 2018). Specific criteria are needed to follow when GEMs are made. Firstly, after the regulation, the strains made should be checked that they are stable after the cloning is done when the protein is formed, which is needed to be expressed. Second, the tolerance level of that exceptionally engineered microorganism should be check for those specific contaminations which are needed to be degraded. Any intolerance can cause the alteration in the microorganism and so that Microorganisms would fail to degrade that particular compounds or contaminant. Third, the particular microbe should also be checked for its survivability in that particular rhizosphere specific to the microbe. These criteria should be considered when GEMs are made to ensure that the GEMs are safe to be released in the environment or any conditions in food substance making (Böttcher & Bornscheuer, 2010). Several approaches are used to generate GEMs. However, there are four main approaches which are made chiefly which include: i. The microbes should be identified before the cloning is to be done in them. These should be checked with the conditions, including their survival conditions like the temperature they can survive, and which nutrients are suitable. Only the microbes which are suited to these conditions should be used. ii. The catabolic pathway of those particular microorganisms in which their breakdown is checked and how they are regulating. If

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possible, specific changes are made in their catabolic pathways to be regulated well in the process. iii. Certain modifications are made in the Microorganisms, like the affinity and specificity towards the Enzyme content in which they are modified, and if needed, their affinity and specificity are also modified. A DNA cluster should be used to enhance the pathway, and so the specificity can be changed. iv. Proper monitoring of the microorganism should be done when making the GEMs and have Biosensors and control to ensure that the GEMs are produced.

These criteria and approaches should be made before and during the production of GEMs to have high efficiency of these microbes and then be used in other fields. The use of GEMs in the field of the food sector to produce the food substances required for many processes in food making as well as in the environment by the process of Bioremediation in which GEMs are used for degradation of specific contaminants are used. 7.6 GENETICALLY ENGINEERED MICROORGANISMS (GEMS) IN FOOD PRODUCTION Throughout human history, Microorganisms have been used in the manufacturing of food products. Even before, knowledge of microbial function in fermentation was known to humans. As microorganisms produce their endogenous enzymes, they helped produce many food products for centuries, like cheese, yogurt, beer, vine, and many more foods. After discovering microorganisms in 1830 and their responsibility in several processes, and after a century with the discovery of the DNA, humans begin methods used to elevate the use of these microbes. The modifications in genetic systems in microorganisms are used to make food products more efficient (Kohl et al., 2015; Authority et al., 2017). Over the past decades, extraordinary achievements in biochemistry and molecular biology have steered the broad application of GEM in the production of medical and food substances, specifically as these processes are progressively viewed as environmentally and animal responsive and gainful. For example, insulin is now formed using microbes rather than pigs, slaughtered for the pancreas, the primary insulin store. Likewise, trypsin and chymosin

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produced by microorganisms can be used instead of collecting trypsin or chymosin from animals such as pigs and cattle. The uses of GEM construction are not restricted to exchanging animal-based manufacturing methods. For example, compared with outdated agrochemicals (such as stevia and vanilla extracts), the GEM invention of the glycosides steviol and vanillin from GEM has many sustainable advantages, including reduced land use and less waste a more stable and affordable supply to meet growing consumer demand. An excellent example of the application of GEM as a technique of producing food constituents is the making of riboflavin which dates back to the 1990s until today, where almost 100% of commercial riboflavin is produced using GEM. Other examples of food elements produced by GEMs today contain vitamins, amino acids, functional proteins (e.g., texture builders), healthy proteins, oligosaccharides, flavors, and sweeteners (Herring & Rao, 2012). Finally, cheese can be made with chymosin (the coagulant enzyme of the milk protein that acts in rennet) produced with GEM in place of collecting the enzyme from the calf’s stomach (Hanlon & Sewalt, 2020). 7.7 COMMON METHOD IN MODIFYING THE MICROORGANISMS USED IN FOOD PRODUCTION Microbes used in the procedure generally have to be processed lest they have demonstrated all of the properties required to produce the economically chosen food substance on a large scale. Therefore, unchanged microorganisms are most common in traditional food developments, such as those used in the profitable manufacture of bread, wine, beer, and yogurt, which are sometimes used nowadays to make sure food enzymes. However, genetic adjustment of a microorganism is often necessary to produce a purely desired substance (for example, if the source organism that naturally produces the substance of interest cannot be cultured on a large scale.) Alternatively, genetic modification can progress effectiveness and decrease the cost of producing an endogenous enzyme or other food substance (Lantz et al., 2010). Specific methods which are used in the modification of the microorganism’s genetic makeup is mentioned in subsections (Figure 7.2).

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FIGURE 7.2

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Genetic modification techniques.

Source: Created with BioRender.com.

7.7.1 NON-TARGETED MUTAGENESIS Before creating modern biotechnology, microbes have been heritably modified for a long time by applying selective stress or random mutagenesis induced with the aid of chemical compounds or ultraviolet radiation. Modification techniques chiefly based on classical desire and mutagenesis are now not the goal due to the fact they introduce DNA in random versions and then extensively select out microorganisms that can extend the production of unique enzymes or preferred phenotypic traits. The remaining genetic makeup of microorganisms produced with the valuable resource of random mutagenesis cannot be predicted in advance, and even presently reachable high-throughput DNA sequencing cannot constantly apprehend all DNA adjustments or their consequences (Derewenda, 2010).

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7.7.2 GENETIC ENGINEERING The primary tool for building GEMs to yield food substances is using in vitro nucleic acid techniques, including genes into selected, robust, and safe microorganisms through rDNA or related technologies, thus imparting functions. The microorganisms produced are said to be GM. This term is different from “genetic modification” from the National Research Council of America 2004 and is used in the terminology of various regulatory guidance documents in the United States and Canada. Scientists first select or develop robust, high-performing host microbes identified as safe (non-pathogenic and do not produce toxins) through genetic engineering. Once an appropriate manifestation in the host is mainly established, molecular methods can be used to insert or delete one or more DNA sequences into the microbial genome, thus enhancing the existing functions of the organism or conferring new functions on it. Types of new or improved functions include production of enzymes or other functional proteins (e.g., u03b1-amylase, lipase, protease, ice structure protein, leghemoglobin) collected from microorganisms for food production, which contribute to the microorganisms produce enzymes from other foods. In addition, ingredients (such as riboflavin, steviol glycosides, or oligosaccharides) make other modifications, such as deleting endogenous genes or inserting transporters, to make microorganisms a more efficient production platform. A sequence containing coding elements in a DNA sequence can express new or improved functions (Makarova et al., 2011). DNA was initially extracted from a microbe and then multiplied and transported to other microbes. Nowadays, artificial DNA sequences are formed by other molecular biology practices introduced into microbes to confer functions. The use of synthetic DNA can very quickly develop GMOs that can produce many modified enzyme proteins that can be verified. In addition, the use of artificial DNA can prevent the involuntary introduction of foreign sequences, such as cloning fragments and even pathogens. The introduction of synthetic DNA permits molecular biologists to reduce the transfer of valuable genes of interest without interaction with pathogens and avoids the relocation of sequences that may encode pathogenicity. Sewalt et al. (2018) detailed the safety considerations of one of these examples, which originated from a potential pathogen from Cytophaga sp. The sequence of \ u03b1-amylase is carefully expressed in Bacillus licheniformis and reported to FDA for its usefulness (Sewalt et al., 2018).

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7.7.3 PROTEIN ENGINEERING Protein engineering is generally used to enhance the function of enzymes. This may be aimed at improving catalytic activity but is most commonly used for custom catalysts. Enzymes will work most efficiently under application situations that may include temperature, pH, or salt concentration, and these conditions are well beyond the optimal range for enzymes (Leisola & Turunen, 2007). For example, baking amylase can be designed to endure a more extended high temperature so that a lower initial enzyme concentration can be used to achieve the same number of catalytic reactions before the enzyme is disabled during the baking process. Initial with endogenous wild-type enzymes, practical protein engineering usually involves genetic engineering of production organisms (significantly when the expression level must also be increased) or gene editing (to test impact) to produce multiple changes. These modifications are verified for their use to produce multiple clones of the improved gene with selected features, and occasionally these matches are combined in consecutive generations to make the most of the improvement. The finally selected mutated enzyme protein may vary from the wild sequence by one or more amino acids. 7.7.4 GENOME EDITING USING CRISPR CRISPR is the most extensively used technology for gene editing. CRISPR is an innovative tool that uses particular proteins stimulated by nature and designed by scientists to promote precise cutting and bonding of DNA. There are three types of gene editing proteins: zinc finger, TALEN, and CRISPR-Cas. CRISPR-Cas has a fashionable design and simple cell delivery. It is now used to treat genetic diseases, produce weather-resistant crops, and improve designed materials, foods, and drugs. CRISPR established a collected palindrome repeating sequence at regular intervals, which periodically repeats DNA fragments in some bacterial sp., as a prehistoric defense system against virus invasion. CRISPR-Cas is a compound made up of enzymes (Cas proteins) and genetic guides (CRISPR sequences), which find and edit DNA together. Scientists use this potent tool (Makarova et al., 2011) by manipulating guide RNA sequences to identify specific DNA codes that represent genetic defects or undesirable traits in any living cell and remove one or more of the DNA codes. Replacement nucleic acid can be inserted at a specific site where the target sequence is disrupted to repair

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DNA, modify the sequence, or introduce new beneficial genes, not just remove the function of the endogenous gene. The use of microorganisms in biotechnology was first defined by Howard Hughes Medical Institute researchers in 2012. Elimination, restoring, or modifying gene functions is known as gene editing (Mao et al., 2013). 7.7.5 AUTHORIZATION AND EVALUATION OF GMO MANUFACTURED FOOD CONSTITUENTS Food producers need to comply with the governing supplies of each country before selling new foods. The addition of GMO-based food in the manufacturing process may not cause significant changes in the structure of food substances, and therefore cannot be interpreted as a novelty in these frameworks. Nevertheless, food producers can still select to evaluate their food constituents through these monitoring procedures to obtain independent confirmation that the use of GEM is measured safe and sees all governing requirements. Any new food substance evaluation includes evaluating the entire production process, including production organisms, fermentation media, equipment, filters, processing aids, and any ingredients in the formulation. The evaluation results of the new food are published in the “EU New Food Joint List.” In the United States, it is more common that the FDA has notified the FDA of a list of substances recognized as safe. The European Union and the United States have completed multiple evaluations of novel foods produced by GEM, including steviol glycosides, oligosaccharides, and a structural protein, but other food ingredients use genetically modified microorganisms (GMMs) (modified by undirected mutagenesis). These include riboflavin and other B vitamins, amino acids such as methionine, and other foods. Most of these foods have become part of the food supply and have been around for more than 10 years, and the use of GEM for production will only represent other novel production processes of the same molecule (Liang et al., 2014). 7.8 GENETICALLY ENGINEERED MICROORGANISMS (GEMS) IN ENVIRONMENT PROCESS BIODEGRADATION 7.8.1 BIODEGRADATION AND BIOREMEDIATION Biodegradation is the naturally catalyzed decrease in the complication of chemical composites certainly; biodegradation is a technique by which

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biological components are broken down into reduced composites through dwelling microorganisms. On completion of biodegradation, the method is known as mineralization. Although, in most instances, the word biodegradation is used to define any or all biologically refereed alteration in a substrate (Fuchs et al., 2011). Bioremediation of pollutants using biodegradation capabilities of microbes encompasses the herbal reduction, though it may additionally be greater via engineered techniques, both with the aid of accumulation of chosen microbes (bioaugmentation) or through biostimulation, in which vitamins are included. Genetic engineering is additionally used to enhance the biodegradation abilities of microbes via GEM. However, many elements are distressing the effectiveness of this technique and the dangers related to the use of GEM (Shan et al., 2013) (Figure 7.3).

FIGURE 7.3

Process of bioremediation or biodegradation through many processes.

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7.8.2 GEMS USE IN BIOREMEDIATION There are four predominant methods to GEM improvement for bioremediation use. It contains: (i) alteration of enzyme specificity and similarity; (ii) pathway development and guideline; (iii) bioprocess change, observing, and regulator; and (iv) bioaffinity bioreporter sensor uses for chemical sensing, toxicity discount, and quit factor analysis. While genetic engineering has formed many lines to destroy otherwise obstinate pollution in a petri dish or a bioreactor, the practical conversion of this lookup into proper in situ bioremediation practices has been very short. The main difficulty in this recognition is the increasing cognizance that most bacterial species often appear in standard enhancement methods are now not performing the bulk of biodegradation in herbal niches and can no longer be suitable bioremediation mediators. The use of secure isotope probing (SIP) and equivalent techniques in microbial ecology have published Pseudomonas, Rhodococcus, and the usual aerobic fast growers extensively appreciated as hosts of biodegradation associated recombinant genes are ways much less considerable underneath herbal conditions. Furthermore, using fast growers as dealers for biodegradation is the inevitable build-up of unwelcome biomass. As an alternative, the top-of-the-line clean-up agent would be the one that shows the most catalytic capacity with a minimal phone mass. The expression of biodegradation genes can be artificially uncoupled from boom using stationary-phase promoters or hunger promoters. In addition, the latest advances in the region of rDNA technologies have cemented the way for hypothesizing “suicidal genetically engineered microorganisms” (S-GEMS) to limit such predictable dangers and to gain well-organized and harmless refinement of polluted websites (Moussavi & Alizadeh, 2010). 7.8.3 SOME DRAWBACKS OF GEMS IN ENVIRONMENT SECTOR The most critical difficulty encountered in profitable bioremediation science affects opposed subject situations for the engineered microbes. Moreover, the molecular purposes are primarily restrained to only a few well-characterized bacteria such as E. coli, P. putida, B. subtilis, etc. Additionally, many bacterial strains want to be tried for creating the engineered microbes. Efforts are made to look at the functioning of modified bacteria about their survival, effortless horizontal gene transfer, which may also affect the indigenous microflora within a complex environmental

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situation. Often the novel scientific research always gives upward shove to nevertheless additional captivating questions about the public concern. In most cases, the bacteria designed for bioremediation approaches have been designed for a unique purpose under laboratory conditions, ignoring the discipline requirement and other complicated situations. However, there is no proof that the deliberate launch of GE microorganisms for Bioremediation has caused a measurable adverse impact on the natural microbial community. At least the overstated idea of hazard appraisal has fueled so much debate and brought about so many research efforts, which have immensely contributed to environmental microbiology. However, the survival of the GE bacteria in complicated environmental conditions is nonetheless a considerable question, desires to be addressed in the light of modern-day findings. Microbial things to do are vital for renewing our surroundings and protecting the world’s carbon cycle. These activities are covered in the time duration of biodegradation. The factors that can be degraded or transformed using microorganisms are many artificial compounds and different chemical elements with ecotoxicological effects like hydrocarbons and heavy metals. However, in most cases, these announcement issues viable degradability estimated in the laboratory with the valuable resource of utilizing chosen cultures and below first-rate boom conditions. Due to a whole range of factors: competition with microorganisms, inadequate supply with quintessential substrates, adverse exterior stipulations (aeration, moisture, pH, temperature), and low bioavailability of the pollutant, biodegradation in herbal stipulations is lesser. So, environmental biotechnology aims to tackle and solve these troubles to allow microorganisms in bioremediation technologies. For this reason, it is vital to resource the activities of the indigenous microorganisms in polluted biotopes and to embellish their degradative skills with the aid of way of bioaugmentation or biostimulation. Genetic engineering is additionally used to beautify the biodegradation abilities of microorganisms. Nevertheless, there are many dangers associated with the use of GEM in the field. However, whether or not such approaches are eventually successful in the Bioremediation of pollution may also make a difference in our potential to decrease waste, eliminate industrial pollution, and experience a more sustainable future. This is because they were using these GMMs to target these specific contaminations in the environment and slowly react with them and completely degrade them. By this, the process of biodegradation is taken place in a very effective manner, and so no

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harm can cause to other substances and other aspects of the environment (Sarayu & Sandhya, 2012). 7.9 CONCLUSION The development of these GEMs has been a significant setback on human history by changing the genetic makeup of the organisms that are readily available in nature so that they are used for specific purposes in many sectors without disturbing their population as these are available in many proportions. Thus, for example, the use of bacteria and yeast such as fungi in the food sector has been a significant role if these Microorganisms in the production of a large number of food substances which are sometimes naturally done or modification of these Microorganisms and introduced them to food to provide certain enzymes that make the food more usable and foods having properties like high nutrient content as well as diseasefree crop and many more qualities in them which can be of high priority in the places where the nutritional deficiencies are seen and therefore can help to control the situation over these places. The use of GEMs in the environment sector has been of great use as these processes of biodegradation produce almost no pollution, and the contaminations which are hardly degraded in nature, such as plastics, are degraded by using these efforts of changing the genetic makeup of the organisms and introduced them in the soil so that the process of Bioremediation and biodegradation takes place in a very efficient way. Furthermore, there are many uses of these GEMs in the health sector. Also, to produce the medicines used in drug therapy are also being used as an effective measure to control various diseases by attacking the specific cells and genes responsible for certain diseases. KEYWORDS • • • • • •

biodegradation clustered randomly interspaced short palindromic repeats genetically engineered microorganisms genetically modified organism polymerase chain reaction transcription activator-like effector nucleases

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Fuchs, G., Boll, M., & Heider, J., (2011). Microbial degradation of aromatic compounds— From one strategy to four. Nature Reviews Microbiology, 9(11), 803–816. Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., & Hasegawa, M., (2009). Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proceedings of the Japan Academy, Series B, 85(8), 348–362. Gaj, T., Gersbach, C. A., & Barbas, III. C. F., (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology, 31(7), 397–405. Gitschier, J., (2009). Wonderful life: An interview with Herb Boyer. PLoS Genet, 5(9), e1000653. Hai, T., Teng, F., Guo, R., Li, W., & Zhou, Q., (2014). One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Research, 24(3), 372–375. Hanlon, P., & Sewalt, V., (2020). GEMs: Genetically engineered microorganisms and the regulatory oversight of their uses in modern food production. Critical Reviews in Food Science and Nutrition, 1–12. Herring, R. J., & Rao, N. C., (2012). On the ‘failure of Bt cotton’: Analyzing a decade of experience. Economic and Political Weekly, 45–53. Hoban, M. D., Lumaquin, D., Kuo, C. Y., Romero, Z., Long, J., Ho, M., Young, C. S., et al., (2016). CRISPR/Cas9-mediated correction of the sickle mutation in human CD34+ cells. Molecular Therapy, 24(9), 1561–1569. Johnson, D. B., Okibe, N., & Hallberg, K. B., (2005). Differentiation and identification of iron-oxidizing acidophilic bacteria using cultivation techniques and amplified ribosomal DNA restriction enzyme analysis. Journal of Microbiological Methods, 60(3), 299–313. Kaji, K., Norrby, K., Paca, A., Mileikovsky, M., Mohseni, P., & Woltjen, K., (2009). Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature, 458(7239), 771–775. Karimi, M., Inzé, D., & Depicker, A., (2002). GATEWAY™ vectors for agrobacteriummediated plant transformation. Trends in Plant Science, 7(5), 193–195. Khan, J., Alotaibi, A., & Deka, M., (2015). Effect of colchicine induced mutation on cellulose enzyme production by Aspergillus fumigatus. World J. Pharmaceutical Res., 4, 461–471. Khan, J., Alshehri, B., & Banawas, S. (2021). Probiotic Potential and Stress Tolerance in Lactobacillus MU1008 Isolated from Chilled Yogurt Samples of Majmaah, IJPSR, 12(1), 654–660. Kohl, C., Frampton, G., Sweet, J., Spök, A., Haddaway, N. R., Wilhelm, R., Unger, S., & Schiemann, J., (2015). Can systematic reviews inform GMO risk assessment and risk management? Frontiers in Bioengineering and Biotechnology, 3, 113. Lantz, S. E., Goedegebuur, F., Hommes, R., Kaper, T., Kelemen, B. R., Mitchinson, C., Wallace, L., et al., (2010). Hypocrea jecorina CEL6A protein engineering. Biotechnology for Biofuels, 3(1), 1–13. Lee, K. Y., Lund, P., Lowe, K., & Dunsmuir, P., (1990). Homologous recombination in plant cells after agrobacterium-mediated transformation. The Plant Cell, 2(5), 415–425. Lei, Y., Guo, X., Liu, Y., Cao, Y., Deng, Y., Chen, X., Cheng, C. H., et al., (2012). Efficient targeted gene disruption in Xenopus embryos using engineered transcription activatorlike effector nucleases (TALENs). Proceedings of the National Academy of Sciences, 109(43), 17484–17489.

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Leisola, M., & Turunen, O., (2007). Protein engineering: Opportunities and challenges. Applied Microbiology and Biotechnology, 75(6), 1225–1232. Li, M., Suzuki, K., Kim, N. Y., Liu, G., & Belmonte, J. C. I., (2014). A cut above the rest: Targeted genome editing technologies in human pluripotent stem cells. Journal of Biological Chemistry, 289(8), 4594–4599. Liang, Z., Zhang, K., Chen, K., & Gao, C., (2014). Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. Journal of Genetics and Genomics, 41(2), 63–68. Lucas, M. S., Dias, A. A., Sampaio, A., Amaral, C., & Peres, J. A., (2007). Degradation of a textile reactive azo dye by a combined chemical-biological process: Fenton’s reagentyeast. Water Research, 41(5), 1103–1109. Makarova, K. S., Haft, D. H., Barrangou, R., Brouns, S. J., Charpentier, E., Horvath, P., Moineau, S., et al., (2011). Evolution and classification of the CRISPR–Cas systems. Nature Reviews Microbiology, 9(6), 467–477. Mao, Y., Zhang, H., Xu, N., Zhang, B., Gou, F., & Zhu, J. K., (2013). Application of the CRISPR–Cas system for efficient genome engineering in plants. Molecular Plant, 6(6), 2008–2011. Martin, D. P., Murrell, B., Khoosal, A., & Muhire, B., (2017). Detecting and analyzing genetic recombination using RDP4. In: Bioinformatics (pp. 433–460). Springer. McFarlane, S. T., (2008). Unthinkable Biotechnology: The Standing-Reserves and Sacrificial Structures of Life Itself. Dept. of English-Simon Fraser University. Meselson, M. S., & Radding, C. M., (1975). A general model for genetic recombination. Proceedings of the National Academy of Sciences, 72(1), 358–361. Morel, V., Fournier, C., Francois, C., Brochot, E., Helle, F., Duverlie, G., & Castelain, S., (2011). Genetic recombination of the hepatitis C virus: Clinical implications. Journal of Viral Hepatitis, 18(2), 77–83. Moussavi, G., & Alizadeh, R., (2010). The integration of ozonation catalyzed with MgO nanocrystals and the biodegradation for the removal of phenol from saline wastewater. Applied Catalysis B: Environmental, 97(1, 2), 160–167. Nicholl, D. S., (2008). An Introduction to Genetic Engineering. Cambridge University Press. Noori, H., & Chen, C., (2003). Applying scenario‐driven strategy to integrate environmental management and product design. Production and Operations Management, 12(3), 353–368. Offringa, R., De Groot, M., Haagsman, H., Does, M., Van, D. E. P., & Hooykaas, P., (1990). Extrachromosomal homologous recombination and gene targeting in plant cells after agrobacterium mediated transformation. The EMBO Journal, 9(10), 3077–3084. Paigen, K., & Petkov, P. M., (2018). PRDM9 and its role in genetic recombination. Trends in Genetics, 34(4), 291–300. Prado, J. R., Segers, G., Voelker, T., Carson, D., Dobert, R., Phillips, J., Cook, K., et al., (2014). Genetically engineered crops: From idea to product. Annual Review of Plant Biology, 65, 769–790. Pray, C., & Huang, J., (2003). The impact of Bt cotton in China. In: The Economic and Environmental Impacts of Agbiotech (pp. 223–242). Springer. Rai, M., & Ingle, A., (2012). Role of nanotechnology in agriculture with special reference to management of insect pests. Applied Microbiology and Biotechnology, 94(2), 287–293.

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Rajasekaran, R., Kirubaharan, J. J., Shilpa, P., & Vidhya, M., (2020). Commonly Used Molecular Cloning Strategies in Construction of Recombinant Viral Vaccines. Poultry Punch, 74–76. Sarayu, K., & Sandhya, S., (2012). Current technologies for biological treatment of textile wastewater–a review. Applied Biochemistry and Biotechnology, 167(3), 645–661. Satterfield, T., Gregory, R., Klain, S., Roberts, M., & Chan, K. M., (2013). Culture, intangibles and metrics in environmental management. Journal of Environmental Management, 117, 103–114. Saxena, G., Kishor, R., Saratale, G. D., & Bharagava, R. N., (2020). Genetically modified organisms (GMOs) and their potential in environmental management: Constraints, prospects, and challenges. In: Bioremediation of Industrial Waste for Environmental Safety (pp. 1–19). Springer. Schurman, R., & Munro, W., (2006). Ideas, thinkers, and social networks: The process of grievance construction in the anti-genetic engineering movement. Theory and Society, 35(1), 1–38. Sewalt, V. J., Reyes, T. F., & Bui, Q., (2018). Safety evaluation of two α-amylase enzyme preparations derived from Bacillus licheniformis expressing an α-amylase gene from Cytophaga species. Regulatory Toxicology and Pharmacology, 98, 140–150. Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., Zhang, K., et al., (2013). Targeted genome modification of crop plants using a CRISPR-Cas system. Nature Biotechnology, 31(8), 686–688. Strauss, B. S., (2016). Biochemical genetics and molecular biology: The contributions of George Beadle and Edward Tatum. Genetics, 203(1), 13. Su, S., Wong, G., Shi, W., Liu, J., Lai, A. C., Zhou, J., Liu, W., et al., (2016). Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Trends in Microbiology, 24(6), 490–502. Sugawara, Y., Iwamori, M., Matsumura, T., Yutani, M., Amatsu, S., & Fujinaga, Y., (2015). Clostridium botulinum type C hemagglutinin affects the morphology and viability of cultured mammalian cells via binding to the ganglioside GM 3. The FEBS Journal, 282(17), 3334–3347. Sun, N., & Zhao, H., (2013). Transcription activator‐like effector nucleases (TALENs): A highly efficient and versatile tool for genome editing. Biotechnology and Bioengineering, 110(7), 1811–1821. Teuscher, P., Grüninger, B., & Ferdinand, N., (2006). Risk management in sustainable supply chain management (SSCM): Lessons learnt from the case of GMO‐free soybeans. Corporate Social Responsibility and Environmental Management, 13(1), 1–10. Tharmalingam, T., Wu, C. H., Callahan, S., & Goudar, C. T., (2015). A framework for real‐time glycosylation monitoring (RT‐GM) in mammalian cell culture. Biotechnology and Bioengineering, 112(6), 1146–1154. Thomson, J., (2001). Horizontal transfer of DNA from GM crops to bacteria and to mammalian cells. Journal of Food Science, 66(2), 188–193. Van Os, H., Andrzejewski, S., Bakker, E., Barrena, I., Bryan, G. J., Caromel, B., Ghareeb, B., et al., (2006). Construction of a 10,000-marker ultra-dense genetic recombination map of potato: Providing a framework for accelerated gene isolation and a genome wide physical map. Genetics, 173(2), 1075–1087.

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CHAPTER 8

Patenting of Living Organisms: Significance, Copyrights, Trade Secrets, and Trademarks MUHAMMAD SAJJAD IQBAL and FAIZA NASIR

Biodiversity Informatics, Genomics, and Post-Harvest Biology, Department of Botany, University of Gujrat, Gujrat, Pakistan

ABSTRACT Patent is a legal document that provides an authority to the inventor/claim holder who could be a design developer, process builder or a new machine manufacturer. It prohibits others for its misuse, selling or making similar types. With the passage of time to protect one’s intellectual property rights (IPRs), patents, trademarks, trade secrets, copyrights, etc., are the types of laws and regulations to comprehend smooth and fair use, profit to the respective bodies and surety to execute safely. Like other things and items, some countries, companies, and scientist use to patent living organisms. Patenting of living forms may be considered as unethical because it tangled with the law of nature and is not acceptable by the society at large. But US Supreme court allows that living organisms can be patent. Plants, animals, and microorganisms of different types are permitted to be patentable. Copyrights are the rights awarded to you by creating an original work, e.g., work reproduction, derivative works, copies distribution, publications, photographs, artwork, etc. Several countries develop their own copyrights to protect owner’s rights, royalties, and privileges. Similarly, trade mark

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represents new logo or sign to identify or distinct product. Trademarks are usually designated with some symbols, signs, diagrams, new creation by combining many small items and representing these. Another important term is trade secret which implies for those documents, recipes, innovations, or discoveries that need to be kept confidential or disclosed with the consent of the discoverer and rights remain with its owner or with company. Trade secret remains valid for a time till it becomes generally public. 8.1 INTRODUCTION Patent refers to the new invention, design, process, variety, and even new machine, etc., and it provides inventor maximum rights. It provides rights only to the owner and restricts anyone else, selling or making an invention for a limited period. Most of the countries adopted strict laws for violation of one’s rights of invention. Patents are not only important for inventors but also for industries as useful for competitive advantages while others do not consider them essential. The World Trade Organization (WTO) TRIPS Agreement, advocates the availability of patents across all of the areas of technology in WTO member countries (Figure 8.1). In general Criteria for patentability are based on novelty, non-obviousness, and industrial applicability.

FIGURE 8.1

IPR representing a number of provisions for innovations.

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The term patent generally implies for the rights that are given to someone who invent something unique, productive, and useful. In some other domain, some kinds of IPR are also designated as patents. For example, the United States affirm industrial designs as industrial patents, plant breeder’s rights, and new unique models including software, chemicals, business methods, and biological are considered as special type of patents for invention. It contains information and analysis which are probably less than currently being sought for patents in particular field such as biomedical (Hassan et al., 2018). Numbers of patents granted per year around the world are shown in Figure 8.2, which elaborate that obtaining patents are now a routine practice and people across the world understands the significance of registering new innovations (Table 8.1).

FIGURE 8.2

TABLE 8.1 1. 2. 3. 4. 5. 6. 7. 8. 9.

Number of patents per year registered worldwide.

List of Top DNA Patent Holders by National Institute of Health, USA

Agribusiness and chemical companies US government (NIH) Public and private universities Pharmaceutical firms Biotechnology firms Genomic technology firms Instrumentation and DNA chip firms Academic research institutions Research hospitals

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8.1.1 RECENT TREND IN PATENTS FOR SARS AND MERS Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the main reason of causing COVID-19, it enters in host cell because of viral protein and cause replication and that replication is attached with SARS-CoV. It can provide insights which will be beneficial for the development of preventive agents and treatment of COVID-19 (Figure 8.3). Figure 8.4 shows the distribution of patents related to SARS and MERS. SARS granted patent number is almost 12 times greater than the MERS (Figure 8.4). Its reason is, maybe the outbreak of SARS accrue round almost one decade earlier. The majority of the patents are related to medicines as 80%, vaccines 35% and 28% for diagnostic agents (Liu et al., 2020). >10, 326 publications are being identified for COVID-19, out of theses WIPO received applications 240, China 616, Europe 189, Japan 179, South Korea 169, and US 599 (Peden & Konski, 2020).

FIGURE 8.3 coronavirus.

Number of patents per year registered and patent applications related to

8.1.2 HISTORY OF PATENTS Historically there was some evidence that Greece firstly granted legal law mechanism in 1474, it was also known as Venetian patent law. While The English patent system evolved into the modern patent system before the beginning of the early middle ages, which recognized intellectual property to drive new one. It became the base for economic activities. In North America, the first patent was awarded to Samuel Winslow in recognition of a new salt making procedure by Massachusetts General Court in 1641.

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FIGURE 8.4 application.

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Number of patents distribution for (A) SARS; and (B) MERS based on

Similarly, few other countries created their own system of patenting like ‘The modern French patent system’ during 1791. On April 10, 1790, US Congress approved 1st patent act whose title was “Promoting the Advancement of Useful Arts.” On July 31, 1790, Samuel Hopkins received 1st patent under this act against the development of a procedure for potash (potassium carbonate). Patent law was revised once in 1793 and then in 1836 with stringent impact. During that time 10,000 patents were approved between 1790 and 1836, and then the practice of granting patents continue throughout the world.

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8.1.3 IMPACT OF PATENTS Patent is an authority which assigns provision to disclose openly, and such rights can be transfer, sell, or withdrawal. It is important that patent owner should not exploit it for personal gains only or any company, as several inventions are improved versions of prior inventions that further in any case owner’s right should be protected and secondly, he/she has the right not to permit someone or restrict its use without its prior approval. 8.1.4 WHY WE HAVE PATENTS? According to Friedrich-Karl Beier’s patents are granted on following grounds: i. for identifying the inventor’s intellectual property; ii. for giving reward to the inventor for his good and useful services; iii. for encouraging scientists, engineers, and technicians to showcase their outcome; and iv. for timely dissemination and access of information and knowledge of that work or product. 8.1.5 BIOTECHNOLOGY AND PATENTS In general, biological processes involving microorganisms, animals, or plants with the help of some technical uses refers as biotechnology. Impact of biotechnology can be seen in health-related medicines, food commodities and agriculture at large. European Union followed the biotechnology guidelines and granted patents in biotechnology since 1998. Likely, Sweden modified their law in May 2004 and provides provision what is allowed to be patent and what is not. Several countries across the globe now have rules and regulations for granting patents for biotechnological entities. India is moving fast in the field of biotechnology and granted several patents for new innovative technologies. Among other items in biotechnology (31%), major contribution is from protein and enzymes (30%), nucleic acid and RNA and fermentation (9%), microorganisms (2%), transgenic (2%), sequencing (5%), vaccines (5%), gene (6%) and bacteria and bacillus (10%) during 1999 to 2003. Whereas a trend is

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provided to show the technologically advanced countries which lead in biotechnology-based patent approval (Figure 8.5). 8.1.6 PATENTABLE BIOTECHNOLOGICAL INVENTIONS Following are the interventions and discoveries that are allowed to be patentable under biotechnology: • Procedures developed, analytical tools and standards including medicinal product; • Biological products like proteins, DNA sequences, microorganisms, and components of human body organs; • A gene with known functions like part of medicinal product or diagnostic tool or isolated part which is associated with some particular function; • Genetically modified organisms (GMOs) or products like microorganisms, animals, and plants.

FIGURE 8.5

Biotechnology patents in the technologically advanced countries.

Source: Reprinted with permission from Huggett & Paisner (2020). © 2020 Springer Nature.

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8.1.7 NON-PATENTABLE BIOTECHNOLOGICAL INVENTIONS Non-patentable biotechnological inventions involve: • Original inventions like fragments and parts of animals, plants or microorganisms which are not further explained; • Plants and animal breeding material are considered as nonpatentable inventions; • Inventions that are against public orders and ethics and society consider them as immoral and unacceptable. For example, patenting of human cloning is impossible as well as against the public ethics. 8.2 PATENTING OF LIVING ORGANISMS When biotechnology developed, it was a DNA and cloning technique that competed with people legally and morally because higher life forms are natural in their existence and no one in the cycle of nature can invent them. The major issues arising in patent life are violations of the “right to life” because the life of man, animal, and plant life is not acceptable to society or law, and it also affects the honor and identity of a natural being. The first idea to patent a drug was considered ridiculous because the drug was for “people” and they owned it. The creativity and intelligence behind the various studies that followed have given companies and humans in medicine and human genes great benefits for their own genes. As time goes on, all previous objections will become unimportant to the benefits of patent genes, such as the idea of treating diseases and synthesizing future life from scratch (Sharma & Jain, 2019). 8.3 CHAKRABARTY’S CASE Ananda Chakrabarty, a scientist at General Electric Research and Development Center, developed a bacterium in 1971 which was able to divide crude oil hydrocarbons into smaller units. Such genetically modified microorganisms (GMMs) have the ability to use as bioremediation of oil spills. In that year, Ananda Chakrabarty applied in PTO for a patent. The claim was refused to grant by the authority. On the basis of the following grounds: (i) as microorganisms are considered as “products of nature;” and (ii) US Congress Section 24 (101) living creatures are non-patent subject of matter.

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8.4 US SUPREME COURT PROCEEDINGS AND AFTERWARDS Chakrabarty claim opens new discussion about possible impact of permits assign for patents concerning GMO, biotech market, patent system and possible future impact on biodiversity as well as food security. Besides this, the ethics of patenting animals and ownership of public-funded research to allow private ownership of inventions raised major concerns. 8.4.1 COURTS DECISION INFLUENCE ON TECH GIANTS It holds a major concern about how much industry benefited and to what extent patents have been granted with respect to GM microorganisms. This needs to see the ideas behind such inventions and their ultimate impact of socioeconomic wellbeing. 8.4.2 COMPREHENSION OF PATENTS AND INNOVATIONS Innovations are the products or produce of new processes, methods, varieties, or breeds that only provide benefits to the inventor when assigned a patent and allows other to use for financial gains. The patent clause Sections I and 8 exhibits the same concept with respect to the implementation of law according to the constitution. Therefore, there is need to analyze comprehensively the complexity of the designs and the aspects that unwind its components. Another aspect is the understanding conceived from both patents and trade secrets with respect to innovation. It is evident that patenting opens new avenues of authenticity of innovation. Before a patent have been granted it passes stringent screening which also involved cost, risks, and longtime usually evaluation and trails and testing. It further, requires value addition and marketing. Marking skills are the major factor which attracts stack holders towards its application. While on the other hand, patent also provide an opportunity of business and quality standards like apple first ever achieve its earning USD 3 trillion. New invention acquires R&D and marketing for new process, product, or design. Likewise, plant varieties, animal breeds and genetically engineered microbes. Thus, new inventions involve money, time, and concentrated efforts to materialize a prototype to design, therefore a small quantity of unique outcomes in the form of an idea or product

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prevails. Here is a point of discrimination exists between high revenue earning firms and small units. In the same context, patent granted unwind related information and knowledge that permits others to develop similar type of products or improved one. This also direct funding sources and inventors to allocate funding on other items. It also stops unavoidable costs for several research groups, and it focuses direction use towards new products or processes. It also provides an opportunity to invest more on technical aspects rather than keep privacy. It is also evidenced that several patents open new door for research and technology-based innovations like the discovery of penicillin by Sir Alexander Fleming, effective against microbes. More than 10 years were invested to refine penicillin in terms of resources and monetary input. Due to importance of II WW and the international struggle which ultimately saved that work. Sir Howard Florey, a Nobel laureate in the Nobel laureate and Fleming for naming penicillin, said that the delay was due to the fact that they did not have a patent on the drug, which he called a major mistake. Some have argued that proprietary patent power can be used to delay new ones. The organization may opt to deny for apply grant of proprietary patent, thereby keeping being unsafe. As far as companies are concerned for the development of patented products, they have their own choice to participate in the developmental program, earn royalty and market it alone or in partnership the mode of business will depend on the provision of rules, honesty, jurisdiction, and copyrights. 8.4.3 INFLUENCE OF COURT’S DECISION ON BIOTECH INDUSTRY Decision in Chakrabarty case provided additional incentives and opportunity to the industry for microbial organisms patenting to commercialize, that would otherwise have been kept secret. 8.4.4 INFLUENCE ON PATENT LAW AND PTO The decision was applauded by an argument that the invention through Chakraborty’s microbe patent was man-made, therefore it is living is no more convincible. Following this, The Patent Office explains it in detail and allows patenting for microorganisms with properties modified by scientists or genetically modified. For example, production of antibiotics

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from microbes from pure culture can be patentable. It solely depends on the laws due to complication, reproducibility, and variation in living organisms’ rather than non-living things. It not only requires proper interpretation but also a description of proper application of the invention. Additionally, it depends how much microorganism covered under patent law, so need for another type of law or act like Plant Protection Act needs to be implemented. It on the other hand becomes too costly and difficult to impose rights properly. Thus, it requires comprehensive reviewing and validation of claims related to living organisms based on novelty and no obviousness. 8.5 CAN ANIMALS AND PLANTS BE PATENT? The answer is yes, modalities should not be restricted to the unique organism or some breed. Development of living organisms through conventional means of genetics and breeding are not included for patents but their products and microorganisms and/or their products modified through genetic engineering or recombinant technologies can be assigned a patent, e.g., GMOs. Therefore, new genetically modified (GM) lines or varieties of animal or plant or microorganisms can be patented like hybrid mice and hybrid seeds. 8.6 THE PROTECTION OF ANIMALS IN JAPAN Animal patents in Japan began after 1988 following assigning patents of animals in the USA. Afterwards several applications were launched, and patents were granted for animal husbandry using old or new biotechnological techniques. 8.7 PATENTING OF TRANSGENIC ANIMALS Patenting of animals have serious concerns not only legally but morally and ethically too. Other life forms have few rights as compared to animals so, they have more reservations. Moreover, the scope and disclosure of animal patents are not clear in several cases. Patents for animals have issue of selfreproduction, which is less concerned in the case of plants. The agricultural industry faces such challenges more frequently as compared to others, wherein it also provides full provision for making transgenic animals which has already allowed for patent. Additionally, animals themselves can now

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be patented, whether in the context of patent law, ‘manufacturing’ and the phrase ‘substance composition’ including transgenic animals. 8.8 PATENTING OF HUMAN ORGANS OR PARTS Nowadays, it is very easy to modify human blueprint by cutting and editing genetic material with the advent of several biotechniques including DNA recombinant technology, gene splicing and genetic manipulation. After awarding patents to animals or microorganisms, several patents have been granted. Patents included are mostly related to procedures, methods or protocols involved in isolation, purification, and gene sequencing. It poses many medico-legal complications with respect to utilization of human body parts involved in experiments and transplantation. Further, importantly totipotent cells of human beings exhibit the capability to develop into a complete human. On ethical and moral grounds, patents of totipotent cells are not allowed because it may create malfunction of certain organs, or it may go beyond certain objectives. 8.9 START OF PATENTS IN JAPAN ON MICROORGANISMS After granting provision of patents to microorganisms in the United States by the decision of the US Supreme Court in “Diamond v. Chakrabarty” case, several patents were awarded in Japan since 1981. The Japan Patent Office (JPO) enlisted “Examination Standard for the Invention of Microorganisms,” made it public that microorganisms are patentable. 8.10 PATENTABILITY OF MICROORGANISMS The group of microorganisms that has been widely exploited by biotechnologists includes bacteria, viruses, and fungi. Biotechnologists convert the genetic material of this microorganism into desirable forms by using different sophisticated techniques of genetic engineering with the help of different enzymes such as restriction endonucleases and ligases. The patenting of living organisms had been excluded from patent law from 200 years ago as these organisms are considered as “products of nature” and are not invented by humans. Before 1980, no patent was given to the living organism. Although patents were originally made for mechanical and chemical process but later on the patent for living entities were included

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in patent law. On January 28, 1873, Louis Pasteur made the first patent based on microorganism which illustrates a process for fermenting beer. He proclaimed that proposed method assists enhanced quality and high quantity of beer from wort after boiling malt extract with other material, yeast will start fermentation (Nair et al., 2010). 8.11 ARE GENES PATENTABLE MATERIAL? Gregor Mendel, who was a monk in Austria, grew pea plants in his garden and studied them to know that how traits transfer from one generation to the next generation. The hereditary unit that Mendel studied is known as “gene.” Genes are basically the constituents of DNA that contain the information about forming a specific protein that performs a specific function in cells. A large part of DNA is known as junk DNA because it does not have any function in the cell. It is very difficult to identify the small portion of DNA that contains information about protein. That portion of DNA is known as coding DNA. Identification of coding DNA or gene is done through sophisticated technology. This whole procedure, isolation, and purification of gene require intensive research. It is due to this work that the patent office issues patent on specific genes. There was a controversy whether the gene should be patentable or not. In U.S. case law, it was well established that the product of natural creatures or their product should not be considered for patent grant. In Diamond v. Chakrabarty case, discovery of naturally occurring plant in the wild is not patentable. But manufacturing a non-naturally occurring subject with human ingenuity can be patented. This means inventions are patentable, but discoveries are not patentable. In the early days, many genes that encoded proteins that can be used for medical purposes were patented. For example, erythropoietin is a protein that stimulates blood cells production. Scientist invents a method to isolate the gene encoding the EPO and putting it into living organism to produce a large amount of it. EPO is used by the anemic patients. Amegen, a company in California obtains a patent for EPO gene. After acquiring the patent, Amegen sued Chugai that was another competing drug company, for using their patented gene. Chugai state that patent for EPO gene is not valid because it does not fulfill the entire statutory requirement for a patent. But the court ruled that the method for isolation, purification, and obtaining the EPO containing DNA is invented by Amegen.

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8.12 INVENTIONS PROTECTION WITH RESPECT TO GENETIC MATERIAL All of the advanced countries are in consensus that discoveries or inventions associated with genetic material, or their products should be patentable and under some laws to provide them protection. 8.13 INVENTION DISCLOSURE RELATED TO GENES AND THE CLAIMS Inventors are entitled to avail reward for their outstanding work and to obtain a patent right within the scope of the disclosed technique. It plays a vital role in to the mechanism in general. As a result, inventions and their patents support industry associated with the development of technology, while it also ensures patent holder rights and third-party reservations. The approach implies in disclosure of the patent details (Figure 8.6). A diagrammatic sketch is well illustrating the disclosure of the invention as it opens new avenues for others and benefits associated with them, even progress or modification in the prototypes or varieties, etc., while monopoly hides several things and restricted ultimate benefits to a limited number of end users.

FIGURE 8.6

Exclusive right commensurate with the disclosure.

8.14 PRACTICES REGARDING PATENTS AWARD 8.14.1 GENES OF HUMAN BEING DNA or genes are the basic unit of hereditary, which are consisted of nucleotide sequences located on chromosomes. Chromosomes direct or encode RNA or/and protein molecules, this phenomenon is nature-based, so nothing new with respect to patent claims. It is a chemical substance

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like others. But patent have been granted to modified encoded genes or proteins, e.g., in case of interferon, granulocyte colony-stimulating factors (GCSF), granulocyte-macrophage colony-stimulating factors (GmCSF), growth hormones, blood coagulating factors, erythropoietin 29 and so on. There is no distinction between human or monkey encoded protein erythropoietin. It covers both genomic as well as the copy DNA (cDNA), with noncoding regions ‘introns’ (Bidfield, 1995). 8.15 AGRICULTURE INTELLECTUAL PROPERTY RIGHTS (IPRS) From last 25 years intellectual property rights (IPRs) relating to food and agriculture has been extended. US Supreme court decision opens the door for patentability of living organisms like plants, animals, and microorganisms. 8.16 PLANT BREEDER’S RIGHTS

Development of new varieties comes under the plant breeder’s right. It provides provision or rights of handling propagation and harvesting of material. 8.17 CRITERIA FOR AGRICULTURE PATENT The main criteria for this are given below: • • • •

Non-obviousness; Novelty; Utility; Reproducibility.

8.18 EXAMPLE OF PATENTED PLANTS • • • • •

Grape plant; Apple tree; Strawberry tree; Apricot tree; Blueberry plant.

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8.19 PLANT FACES BIOPIRACY CASES • • •

The Neem tree; The Enola bean; Basmati rice.

8.20 BASMATI RICE PATENT CASE Basmati rice is the recognition of Pakistan and India throughout the world for export of unique aroma-based rice. In 1997, US PTO granted a patent to RiceTec Incorporation to call the aromatic rice grown outside India “Basmati,” both India and Pakistan showed serious concern and requested to revert the decision. Because it had serious concerns related to trade business across the world. It was further, describes that the grant of patent to RiceTec violated the Geographical Indications Act under the TRIPS agreement. After hearings, it was decided to named basmati rice 867, with novelty as “Basmati Rice Lines and Grains” providing RiceTec exclusive rights to any basmati hybrid grown anywhere in the western hemisphere. 8.21 PATENT ADVANTAGES TO RICETEC The advantages are following: • • •

Proprietary rights to the crosses and seeds; It helps to capture US market; Using terminology Basmati.

The main advantage is that it is not known as aromatic rice in the United States but provide provision to label Basmati for export purposes. 8.22 ADVANTAGES OF PATENTING LIVING ORGANISMS Organisms can reproduce themselves, therefore they can develop new species or strain or offspring for particular means, and for example production of human insulin is an expensive and time taken procedure. But due to the approval of a patent related to insulin production through evolutionary processes it becomes benefited. Another advantage is that usage and product development from microorganisms become secured due to patenting, and everyone can get benefits of such invention. Patents designated for microorganisms dominated commercially due to royalty

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which needs to be paid accordingly. It is assumed that generally one patent one organism is usually preferred for mass scale production rather exploitation for multiple outcomes. It can be concluded that that an inventor invents ad patented its best product and its use. 8.23 COPYRIGHTS It can be defined as “creation of genuine work and product which assign some rights that how to use it called copyrights.” It includes but is not limited to the use of reproduction of original work, copies, prints, photographs, and historical events, etc., for general public and public performance. For example, an author or teams of authors, or publisher or product designer have the authority to transfer it or public it. He or she has the sole rights and even can get benefits for years ahead. Violation of copyright has plenty and one can claim his or her work with sole distributor rights. License holders can share work or product of someone with its originality and full rights so it can be a safety against piracy. Several countries have their own system of patent and copyrights, trademarks, and trade secrets. Copyright Act Title 17 is used for the protection of copyrights in the United States. Copyrights protect the original and legal work of authorship. Such considerations include databases, computer codes, books, music, sound recordings, architectural works, and paintings, etc. Copyrights have some uniqueness as these are different from patents no such industrial application or novel idea (Campi & Duena, 2019). Copyrights assigned to someone means imposed o persons, companies, and marketing agencies, unless or until copyright or agreement have any mutual undertaking for some specific time period. Often, it happens that job nature or working with some other firm may automatically lose copyright status and ultimately ownership (Poticha & Duncan; 2019). Further, copy right provides publishers and companies protection of their documents and pictures or photographs, etc., legal rights and claims which others could not reproduce without permission. 8.24 TRADE SECRETS Trade secrets are usually assigned to those ideas which are unique and market capturing understanding for probable excel or profit (Figure 8.7). It could be information, formulae, some style, instrument, methodology, or

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key steps (Ettredge et al., 2018). It must be kept secret by the owner as long as someone desires. Trade secrets are independent from protection as patents. Protection comes when a new idea of trade secret generates, hence no formal procedure for application or registration of trade secrets. It may be protected as longer as it remains secret unless generally open. While in the case of the patent, the information and data always need to be shared and disclosed to the patent granting agencies. Further it is evident that scope of the trade secrets is different from patents, patents prohibited all possible uses from others while in case of trade secrets law only restricted acquisition and misappropriation by improper means like theft. There are several options for IPRs to protect one’s invention or copy right or trade secrets, although most of the focus was on patents registration in the past (Hall et al., 2014). It is obvious that trade secrets provide an alternative to patents for possible means of protection of proprietary information. Percentage of obtaining trade secret firms are gradually increasing per year basis across the world that might be due to benefits associated with the working in freer environment as compared to the patents (Figure 8.8).

FIGURE 8.7 Trade secrets applying various means of business tactics.

The National Conference of Commissioners on Uniform State Laws (1985, p. 5) defines a trade secret as provided below after advocating by Uniform Trade Secrets Act (UTSA) (Ettredge et al., 2018): • Economic benefits acquiring by the owner, which are not disclosed to others; • All possible struggle to keep them secret.

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Patents have legal protection therefore more secure, but trade secrets have some advantages as there is no need to disclose technical specification, hence considered more secure (Jensen & Webster, 2009).

FIGURE 8.8

Percentage of trade secret firms per year.

8.25 TRADEMARKS It can be defined as “a sign, symbol, picture, model that indicates and differentiate products of a group or industry or organization from others in the market.” 8.26 HOW IT WORKS? Trademarks Ordinance (2001) provides means of IPRs and protection by registering logos, signs, or trade symbols, at large. Moreover, it provides awareness especially among business communities to protect and service in fairways. It provides full protection that one cannot exploit and use the brand name or its images. It is just a logo or symbol that allowed businesses legal right and not a physical asset. In the United States PTO registered trademarks under common law which gives legal protection, although obtaining trademarks is somewhat expensive as patents. 8.27 SIGNIFICANCE OF TRADEMARKS Generally, trademarks are important for brand publicity, therefore these are not for disclosing financial reports, but it values an asset (Figure 8.9).

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It rests with a company if it buys shares or assets of another company or products, then trademarks or specific logo of the company in profit may excel in market well than other one. Value of the product and trademark varies from market to market. Likewise, several trademarks are unable to catch market while some become more and more popular, and some lose in spite of heavy investment.

FIGURE 8.9 An illustration of trademarks representing logos.

8.28 COMPARISON OF TRADE SECRET AND PATENT PROTECTION Although patenting of microorganisms’ exhibit advantages but a company may decide to keep trade secrecy. Following parameters need consideration which selecting between both options: An organism or its product could be commercialized. Is there any requirement remaining for legal patenting grant? Still there are opportunities for others to rediscover it. Is this patent is genuine invention, what would be the ultimate benefits and its commercial application? • Can others have the capacity to make amendment in it if information related to patents is disclosed? • Would there be a publication based on this patent and the cost? • • • •

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First two points provide ample opportunity to decide whether patenting will be good or just trade secret enough. But of course, patent provide ultimate production as in the case of Chakrabarty invention. In contrary, last two points indicate mild nature of ideas and there and their market. While publishing about experimentation or invention will results no more secrecy of the idea and product. The inventor as well as company may decide to choose for publication experimentation or product or may choose to keep trade secret depending on its cost and its impact. 8.29 PATENT OFFICES 8.29.1 WHAT DOES PATENT OFFICES DO? Patent offices throughout the world used for the meaning of granting patents related to new inventions, whether applied by governmental organizations, scientific organizations, or the general public. Patent offices or organizations are usually established under governmental act approved by constitutional institutes. This office has the right to accept the application and grant a patent or assign patent number after registration on some defined parameters of have the right to decline the request (Competition, 2008). One can submit an application for grant of patent in his/her own country or outside the country. 8.29.2 MAJOR PATENT AWARDING ORGANIZATIONS Several major patent offices are working throughout the world like US Patent and Trademark Office (USPTO), European Patent Office (EPO), Japan Patent Office (JPO), Korean Intellectual Property Office (KIPO), China National Intellectual Property Administration (CNIPA formerly SIPO) in China, Intellectual Property Organization of Pakistan (IPO) and Indian Patent Office (IPO), etc. 8.29.3 PATENT OFFICE EXAMINATION The patent system is designed to encourage investment in innovative activities to provide investors with legal means to get a fair return on their

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investment (Figure 8.10). Not all inventions are patented, however, and patent office’s conduct examinations to distinguish between those that are novel and inventive should be patented and those that are not, therefore should not be patented (Jensen et al., 2008).

FIGURE 8.10

Patent office examination flowcharts.

KEYWORDS • • • • • •

Food and Drug Administration genetically modified organisms polymerase chain reaction restriction enzymes secure isotope probing suicidal genetically engineered microorganisms

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REFERENCES Barton, J. H., (1991). Patenting life. Scientific American, 264(3), 40–47. Bidfield, G., (1995). In: Vogel, F., & Grunwald, R., (eds.), Patenting of Human Genes and Living Organisms (p. 244). Springer-Verlag. ISBN: 3 540 58148 0. Genetics Research, 66(3), 277–278. Campi, M., & Dueñas, M., (2019). Intellectual property rights, trade agreements, and international trade. Research Policy, 48(3), 531–545. Competition, D. G., (2008). Pharmaceutical sector inquiry. Preliminary Report, 28. Ettredge, M., Guo, F., & Li, Y., (2018). Trade secrets and cyber security breaches. Journal of Accounting and Public Policy, 37(6), 564–585. Hall, B., Helmers, C., Rogers, M., & Sena, V., (2014). The choice between formal and informal intellectual property: A review. Journal of Economic Literature, 52(2), 375–423. Hassan, M. M., Zaveri, A., & Lehmann, J., (2018). A linked open data representation of patents registered in the US from 2005–2017. Scientific Data, 5(1), 1–9. Huggett, B., & Paisner, K., (2020). Biotech patenting 2019. Nature Biotechnology, 38, 921, 922. Jensen, P. H., & Webster, E., (2009). Knowledge management: Does capture impede creation? Indus. Corporate Change, 18(4), 701–727. Jensen, P. H., Palangkaraya, A., & Webster, E., (2008). Application Pendency Times and Outcomes Across Four Patent Offices. Melbourne Institute of Applied Economic and Social Research, University of Melbourne. Kevles, D., (2002). A History of Patenting Life in the United States with Comparative Attention to Europe and Canada: A Report to the European Group on Ethics in Science and New Technologies. Office for Official Publications of the European Communities. Liu, C., Zhou, Q., Li, Y., Garner, L. V., Watkins, S. P., Carter, L. J., & Albaiu, D., (2020). Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases. 6(3), 315–331. Nair, R. B., & Ramachandranna, P. C., (2010). Patenting of microorganisms: Systems and concerns. Journal of Commercial Biotechnology, 16(4), 337–347. Peden, A. S., & Konski, A. F., (2020). Coronavirus Innovation Guideposts on the Eve of the COVID-19 Pandemic. Blog. Personalized Medicine Bulletin Coronavirus Resource Center: Back to Business. Poticha, D., & Duncan, M. W., (2019). Intellectual property—The Foundation of Innovation: A scientist’s guide to intellectual property. Journal of Mass Spectrometry, 54(3), 288–300. Sharma, A., & Jain, R., (2019). Patenting: Product of Nature. International Journal of Legal Science and Innovation, 1(2), 10. Wagner, R. P., (2009). Understanding patent-quality mechanisms. University of Pennsylvania Law Review, 157(6), 2135–2173.

CHAPTER 9

Negative Impact of Recombinant DNA Technology on Life TWINKLE DIXIT,1 NAMRATA DUTTA,2 and ARPIT SHUKLA3

Department of Microbiology and Biotechnology, Gujarat University, Ahmedabad, Gujarat, India

1

Deparment of Microbiology, Swarrnim Startup and Innovation University, Adalaj Kalol Highway, Gandhinagar, Gujarat, India

2

Department of Biological Sciences and Biotechnology, Institute of Advanced Research, University of Innovation, Koba Institutional Area, Gandhinagar, Gujarat, India

3

ABSTRACT Recombinant DNA technology was only a theory a century ago when it came to improving desired traits in live organisms by regulating the expression of target genes. However, in more recent times, this domain has proven to have significant effects in advancing human existence. Scientists utilize rDNA technology for a variety of objectives due to its broad range of uses, but there are several hazards involved in doing so. Recombinant technology may negatively impact the environment, human health, and animals, either explicitly or implicitly, based on a study of significant findings. Additionally, the increased use of this technology has raised safety issues including gene pollution of the environment resulting

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in superweeds and antibiotic-resistant microbes. Increased use of genetically modified foods has been linked to a number of health problems in both humans and animals, including toxicity, antinutritional effects, allergenicity, and carcinogenicity. Due to the potential for harm to both the environment and human life, several safety precautions must be taken when utilizing this technology. This chapter describes in great depth a few negative consequences of recombinant DNA technology. 9.1 INTRODUCTION The goal of this chapter is to depict and communicate some of the potentially harmful effects of recombinant DNA technology (RDT) on the environment, human health, microbes, plants, and animals. Boyer and Cohen performed the first recombinant DNA (rDNA) experiment in 1973 (Cohen et al., 1973), in which identical restriction enzymes (REs) were employed to digest DNA from two different sources before splicing the resultant DNA pieces together. This type of therapy enables the separation and modification of genes, as well as the formation of different DNA molecules and creatures. Genetic alterations carried out by rDNA techniques can be developed to a great extent more rapidly and exclusively than those induced by other traditional methods. rDNA technology may be found in a range of different industries, including medicine, pharmaceutics, agriculture, the environment, food, and cattle breeding (Berkel et al., 2002; Clemente et al., 2001; Gucukoglu et al., 2006; Saltik et al., 2010). RDT has provided the momentum for the expansion of hundreds of companies, globally, that hope to beneficially exploit this technology. With the rapid rate of advancement in this industry, it is critical that potential risks and hazards, both to humans and to the environment, be analyzed and addressed in a purposeful and timely manner. As with the employment of any technology, there are hazards associated with the expansion of biotechnology and the use of its products, both in terms of health and the environment. One cannot predict the impact of GEOs in a new environment at this time because no past or precise data base exists on the actions of genetically modified organisms (GMOs) in the environment, and no basic method for determining the outcome of such an introduction currently exists. The impact of a large-scale release of genetically engineered microorganisms into the environment is difficult to predict since, unlike higher animals

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and plants, they reproduce swiftly and could move considerable distances. Furthermore, their prospective interactions with other creatures in the ecosystem are not well known (Tangley et al., 1983). Figure 9.1 depicts an outline showing biohazards of rDNA technology on environment, human, and animal health.

FIGURE 9.1 Hazardous impact of rDNA technology on environment, human, and animal health.

9.2 HARMFUL EFFECT OF RDNA ON ENVIRONMENT Genetically engineered creatures have become the subject of heated disputes, which have alarmed a considerable portion of society. Genetic engineering technique produced new organisms that could present an ecological problem. The effects of a genetically altered creature on the ecosystem cannot be foreseen. New genetically engineered species discharge can lead to imbalance in the ecosystem of a province. Any mistake in creating the genetics of a bacteria or virus, for example, might result in a stronger variety that, if unleashed, could create a devastating epidemic. This could be lethal in human genetic engineering creating troubles ranging from slight medical problems to death (Mercer et al., 1999). This is because of recent advances in the domains of genetics and molecular biology. Prior to discussing the crucial GMOs, commonly

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known as GMOs for short, some background information on biotechnology is useful. Despite being an economically essential input, GMO-containing goods offer a variety of benefits and drawbacks. The key point of contention for those who support the usage and expansion of GMO-containing goods in discussions is that GMOs produce longer-lasting plants that can withstand harsher circumstances while maintaining high quality at a lesser cost. Reduced pesticide consumption due to the use of GMOs, which also makes it potential to transfer genes giving resistance against plant diseases and pests. It is also likely to cultivate plants to be resistant to drought and other inconsiderate climate conditions. GMO-containing plants can be used to remediate hazardous heavy metal-contaminated soils and surface waterways (Atsan et al., 2008; Berkel et al., 2002; SPO et al., 2000; Gucukoglu et al., 2006). The fact that GMOs have risks and disadvantages in addition to their advantages, as the biotechnology practices are quite new and there is limited information about its possible harmful effects, serious uncertainties have been aroused about in the long run. The leading dispute against is the gene escape. Considered as the main threat of GMOs against the environment, gene escape is the contamination of pollens of genetically modified (GM) plants to other plants of the identical or like species or to the wild plant forms by means of outside factors including insects or wind (Berkel et al., 2002; Demir et al., 2007; James et al., 2005). Genes transmitted to plant species for a specific reason infect other wild species as a result of gene escape, resulting in a significant decline in biodiversity in the long term by causing these species to lose their genetic traits (Kaynar et al., 2009; Magnusson et al., 2002). Another concern posed by GM plants is the likelihood that agricultural pests would undergo genetic mutation in order to become more resistant to genes created over time. It should be emphasized that gene transfer can result in resistance failure in relevant plants. Furthermore, transgenes put into agricultural goods can change the nutritional qualities of these agricultural products by boosting some nutritional values while reducing others. Another critical concern is that some lethal microbes or super plants may emerge during biotechnology studies and field tests; these species may become free if stringent precautions are not implemented, putting environmental balance and human health at risk. As a consequence of advancements in the domains of molecular biology and genetics, GMOs have been the subject of many heated discussions that affect the majority of society. It is a well-known truth that expertise is essential in generating an accurate assessment of

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the benefits and drawbacks of GMOs, limiting possible dangers, and responding cautiously. Gene flow is considered a most important evolutionary force which brings changes in gene frequencies beside with mutation, genetic drift, and selection (Lu & Yang et al., 2009). Gene flow can concern the environment by creating a decrease of differentiation between populations as well as an enhancement in diversity between individuals within a population (Mertens et al., 2008). The structure of genetic diversity (GD) is also one of the outcome of gene flow (Gepts & Papa, 2003). The introduction of non-native GMOs in the ecosystems make possible long-standing risks to the environment and it is somewhat not easy to predict their results. Scientists from several fields throughout the world are concerned about the possibility of transgenic sequences being transferred to related wild species or weeds via recombination or lateral gene transfer. Effects of gene flow also require to be addressed jointly with special effects on nontarget species, species displacement and destruction, disturbance in soil micro-environment and species of ecological distress and biodiversity disturbance (Layton et al., 2015). The likelihood of evolution of novel species cannot be ignored and could also guide to an infinite number of biotic connections (Beusmann & Stirn, 2001). Naturally, plants make use of toxins to protect themselves against threats like pests and pathogens. Such chemicals cause toxicity to abiotic and biotic factors of the environment. Toxins such as glycoalkaloids, delta endotoxins and ricin are of elevated risk concerns and are largely investigated. Bt delta endotoxins have been targeted in most GM plants and the outcome of their proteins on the environment as well as friendly organisms have been studied widely (Yu et al., 2011). Among various sources of transgenes, the most common studied are bacteria other than that animals, fungi, humans, and plants have also been used as sources of various transgenes. Direct gene transfer expresses the preferred proteins in the recipient organism while through molecular breeding frequent parental genes are split and reassembled to express novel proteins which are not there in nature. For example, a new carotenoid was articulated in Escherichia coli (E. coli) by shuffling of DNA coding for a pair of enzymes concerned in carotenoid biosynthesis pathway (Schmidt-Dannert et al., 2000). So, there exist risks attached with natural as well as new toxins being articulated in the plant body. Risk distress of natural toxins might be based on certain developed models. New toxins may have both target and

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non-target effects on life. We are concerned with risks from both types of toxins, either natural or new. Engineered toxins responsible for growth or stress resistance could have astonishing effects on the ecosystem through negative connections. 9.2.1 EFFECTS OF RDNA TECHNOLOGY ON SOIL Continuous discussion is still going on among scientists and farming communities about the effects of GM crop introduction on ground water and water reservoirs. This debate is closely tied to the extent and quantity of usage of herbicide on GM crops. As it is known, GM crops are tolerant to herbicides and invite wide-ranging herbicide applications (Benbrook, 2012). This rise in herbicide use was not direct, i.e., substitution of more toxic herbicide which persists more in the environment with glyphosate (Duke et al., 2012). In a logic, there is a decrease in the application of tones of toxic herbicides, and a rise in glyphosate-based herbicides is observed (Benbrook, 2016). The most broadly used herbicide in the world is Glyphosate. Glyphosate can reach the soil from the direct interception of spray for the duration of early season or post-harvest applications, from overflow or leaching of the herbicide from vegetation and by exudation from roots or death and decay of plant material (Duke et al., 2012; Kremer et al., 2005). The adding up of glyphosate in farmland water and finally to the aquatic ecosystems and its impact on aquatic life is evident. The antimicrobial activity of glyphosate is an issue of discussion also, because big scale applications of glyphosate would definitely upset microbial communities at farm scale (Samsel & Seneff, 2013). Concurrently, transport of Bt toxins from GM crops to soil and water have many likely routes including pollen deposition during anthesis, root exudates, and GM plant residues (Yu et al., 2011). Data exists that Bt toxins combine to the clay and humic substances, rendering the proteins biodegradable (Clark et al., 2005; Saxena & Stotzky, 2000). Once the protein is attached to the clay particles their sensitivity to degradation decreases as observed by (Stotzky et al., 2004) with particular reference to Cry1Ab, Cry1Ac, and Cry3A in root exudates of GM maize, canola, potato, cotton, and rice. But the accidental effects of these proteins on soil residing organisms were not constant and were not used by roots of non-GM plants. Insignificant pH levels under Cry1Fa2 GM maize were viewed as compared to soils

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under non-GM maize statistically (Liu et al., 2010). A vast number of studies have revealed that Bt proteins from transgenic plants broke down quickly after entering the soil and that only a small quantity of them may persist for an extended length of time, implying that Bt proteins do not bio-accumulate in soil (Rauschen et al., 2008; Yu et al., 2011). Moreover, the existence of Bt toxins in soil is mostly determined by the kind of toxin and soil, rather than the number of articulated transgenes (Rauschen et al., 2008). There are proven health benefits for agricultural workers in China (Pray et al., 2001) and South Africa as a result of fewer chemical pesticides being sprayed on cotton (Bennett et al., 2003). 9.2.2 EFFECTS OF RDNA TECHNOLOGY ON BIODIVERSITY Extensive profitable cultivation of GM crops particularly herbicidetolerant crops create severe problems to the ecology density and decrease in biodiversity. In addition to production loss and contamination, weeds are eco-friendly in other ways; consider the reduction of soil erosion caused by weeds and the availability of habitat for a variety of beneficial creatures (Mertens, 2008). However, research revealed that the density, variety, and GM technologies clearly reduced the biomass of agricultural seed banks compared to traditional systems (Bohan et al., 2005). Farm scale evaluations (FSE) in the United Kingdom (UK) revealed a 20–36% drop in weed seed bank (Andow et al., 2003). However, the report revealed that dicot weeds were more vulnerable as compared to monocots. Fast changes in habitat damage will largely impact changes in food webs and food supplies. So, the equilibrium of the predator-prey systems becomes even more vital in addition to the impact on favorable organisms. This will not stop here, of course, troubled tri-trophic interactions and symbiotic associations will also be the end result leading to complex interruption in the food web. It is understandable that such interruption in weed, insect, and pest management will, in turn, end up with better use of pesticides. This amends in resource accessibility, cause secondary effects on advanced trophic levels in most cases. Another potential outcome is a shift in the food chain (e.g., from herbivore to detritivore). In farmlands where glyphosate-tolerant soybean and maize were planted, a short-term change in soil biota was detected. Different kinds of herbicides and insecticides used, timescale of herbicide or

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insecticide usage, target crop, rotational, and agronomic practices used, local fauna and flora, management history, alterative hosts for friendly insects, surrounding habitats, and microclimatic conditions are all factors in the degree of adoption, frequency of application, and disturbance of farmland biodiversity (Mertens, 2008). Bt crops, like herbicide-tolerant GM crops, have been debated for their possible hazards to biodiversity. Pesticides are frequently transported beyond crop fields and can show significant impacts on aquatic and terrestrial ecosystems or on plant populations in the locality of crop fields. Birds and mammals are the most significant targets, and several studies have found little or no evidence of Bt toxicity in these creatures (Flachowsky et al., 2005b; Aris & Leblanc, 2011). In a broader sense, the promotion of HR GM crops has a negative impact on biodiversity (Bohan et al., 2005; Lovei et al., 2013). The highlighted threats to biodiversity are likely to be recognized throughout time, and hazards must not be overlooked. However, one short-term food web evaluation (a two-year investigation) in reaction to the development of GM maize discovered the occurrence of stable and complex food webs and their persistence was not compromised. The study included GM maize having resistance against, Lepidoptera, Coleoptera, and glyphosate and mostly focused on arthropod food webs with an experimental population of 2,43,896 individuals (Szenasi et al., 2014). 9.2.3 HARMFUL EFFECT OF RDNA ON HUMAN HEALTH There is debate going on regarding the effects of GM products on human health. The presence of undiscovered allergens in the GM food supply poses the greatest risk to health. There is no indication that humans who react to allergies would react similarly when the allergen is introduced into other creatures. For example, a previous study found that those who are sensitive to or allergic to nuts exhibited a response to GM soybeans that had a Brazil nut protein put into them (Nordlee et al., 1996). On the whole, there is a still slight proof that GM crops create a major health hazard to consumers. It was recently concluded by the Centers for Disease Control in which it found no association between a claimed allergic reactions and processed food that consists of a GM product. However still GM agricultural products might lead to major risks for human health. Various studies have shown that using GMO goods might cause antibiotic resistance

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in the human body, as well as allergic and toxic responses. Genetically Engineered crops are not a natural food for both the human and animals.

Its genetically altered characteristics for its resistance against to definite

virus, pest, and insect may have irregular danger both in the human health and to minor form of life in the biodiversity directly or indirectly. The intake of Genetically Engineered foods (horizontal gene transfer) may lead to digestive disorders which can lead to numerous health problems. Therefore, it can create abnormal trouble in the human microbiome. It is also observed that GM products have carcinogenic impacts (Atsan et al., 2008; Clemente et al., 2001; Demir et al., 2007). Glyphosphate, the most commonly used weed killer in the world is particularly used in the production of the majority of genetically modified, or GMO crops. Glyphosphate was originally launched in the mid-1970s, and its use has steadily risen since then, reaching a high in 2005 (Ciati et al., 2020). Its impact on human health was not very severe at the start. Since many years data of a continuous harmful impact of this pesticide on humans has been observed. However, Government health control authorities (both in the United States and Europe) continue to allow Glyphosphate use because of the proof provided by the Glyphosphate company. In 2015, the International Agency for Research on Cancer (IARC) concluded that Glyphosphate is likely to be the second most carcinogenic (Class 2A) in terms of risk. The usage of GE crops has one significant drawback: it may result in “abnormal” mutations in a plant’s own naturally occurring proteins or metabolic pathways, leading to the sudden production of toxins or allergies in food (Bohn et al., 2014). 9.2.4 IMPACT ON HUMAN HEALTH BY USE OF VIRAL DNA IN PLANTS The cauliflower mosaic virus 35S promoter (CaMV35S) is used by the majority of crops to change the imported gene. There has been some debate over whether the highly infectious CaMV35S virus may be horizontally transmitted and cause illness, mutagenesis, carcinogenesis, dormant viral reactivation, and the formation of new viruses (Hodgson et al., 2000). It was discovered that the CaMV35S promoter transcribed transgenes transient expression in mammalian cells. As a result, the likelihood of genes controlled by the 35S promoter being expressed in

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animals increased (Tepfer et al., 2004). In one study, however, scientists were unable to identify DNA transfer in mice or CaMV35S transcriptional activity using real-time polymerase chain reaction (PCR), however despite these findings, they underlined the need for more research (Paparini & Romano-Spica, 2006). CaMV in typical diets, according to (Ho et al., 2000), is not extremely infectious and mammals were not able to digest it. In other reported data, it was stated that humans have been ingesting CaMV and its 35S promoter at elevated levels, but despite of this it never recombined with human viruses or shown to cause any disease in humans (Paparini & Romano-Spica, 2004). 9.2.5 ANTIBIOTIC RESISTANT GENES TRANSFERRED TO BACTERIA IN THE GASTROINTESTINAL TRACT Antibiotic resistance genes used as markers in transgenic crops may be passed on to pathogenic gut bacteria, resulting in a decrease in antimicrobial treatment efficiency, which is a hot subject these days. A 26-day research was undertaken to investigate the allergenicity and toxicity of GFP in male rats, and it was established that GFP has very minimal allergenicity risk (Richards et al., 2003). Only one transgenic plant (canola) having GFP has been tested for toxicity. Because GM will be ingested by people for an extended length of time, it is critical that the marker gene of every transgenic organism be assessed for toxicity in long-term research. One major problem associated with GM foods is the probability that genes introduced into the plant might be absorbed by the gut and become involved in the genetic make-up of consumers. In numerous studies it was reported that cows, mice, and chicken that had been fed by GM soybean and corn respectively were containing short DNA fragments of GM plants (Hohlweg & Doerfler, 2001; Phipps & Beever, 2002; Phipps et al., 2003). In addition, fragments of recombinant cry1Ab gene were found in the gastrointestinal tract but not in the blood of pigs fed with Bacillus thuringiensis (Bt)11 corn (Chowdhury et al., 2003). Digestion process does not break down the DNA fragments therefore fragments were observed in the gastrointestinal tract. PCR can detect the fragments of transgenic genes in tissues of animals but not in blood because they are in very low levels in circulation and certain other susceptible methods of detection are required. BT endotoxin, Cry9C produced by modifying

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a variety of genetically engineered corn may cause potentially dangerous immunological responses such as allergic hypersensitivity (Bernstein et al., 2003). “Antifreeze” protein produced by GM yeast isa protein resulting from fish is being taken into account for use in foods such as ice creams. Fish allergies are extremely prevalent, and such proteins may pose a risk to sensitive humans, despite the fact that the sole clinical investigation looking into this possibility found that it does not develop allergenicity (Crevel et al., 2007). 9.2.6 POSSIBLE ASSIMILATION OF GENES INTRODUCED IN A GM PLANT FROM THE GUT One major problem associated with GM foods is the probability that genes introduced into the plant can be absorbed by the gut, and due to this absorption, they can become part of the genetic composition of consumers. In one investigation, researchers were unable to find glyphosate-resistant fragments in tissue samples from pigs fed glyphosate-tolerant soybeans, as well as transgenic and indigenous plant DNA in chicken breast muscle (Jennings et al., 2003a, b). In contrast to this, in another study, orally administered naked M13 phage DNA was observed in the mice blood (Schubert et al., 1994). Furthermore, in cows, mice tissues and in chicken that had been fed GM soyabean and corn respectively short DNA fragments of GM plants have been observed in their white blood cells (Beever & Kemp, 2000; Einspainer et al., 2001; Phipps & Beever, 2001; Hohlweg & Doerfler, 2001). Furthermore, recombinant cry1Ab gene fragments were found in the gastrointestinal tract of Bt11 corn-fed pigs but not in their blood. Hence it is quite evident from this that small fragments of DNA are not broken down completely by the digestion process (Chowdhury et al., 2003). However, these DNA fragments are not observed in the blood of animals by PCR because they are at quite low levels in circulation. Thus, more sensitive, and sophisticated methods are needed for detection (Pusztai et al., 2003). According to one study done by Flachowsky et al. (2005), it was observed that GM DNA which is introduced into cells of the gastrointestinal tract will usually have no biological adverse effects because the DNA will be degraded in the cell.

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9.2.7 HARMFUL IMPACT OF RDNA TECHNOLOGY ON ANIMALS Ewen and Pusztai were the scientist who first reported that it is necessary to cautiously test GM plant product on animal models (Ewen & Pusztai, 1999). The bulk of GM foods have been studied in animals, and the disputed data has been reevaluated and published by Monsanto’s 90-day feeding study on GM maize. Mon863 (Seralini et al., 2007). Even while prolonged feeding of excessive quantities of particular “foods” to animals might result in nutritional imbalance, it should be confirmed that this is the single method for any substance to demonstrate toxicity (Varzakas et al., 2007). 9.2.8 EFFECTS OF GM FOOD ON THE GASTROINTESTINAL TRACT OF ANIMALS Disturbance, swelling, multinucleation, and increased degradation of ileal surface cells in rats has been previously reported due to consumption of GM potatoes expressing Bt-toxin. Thus, health can be adversely affected by consumption of GM foods (Fares and El-Sayed et al., 1998). In rats fed with Flavr-Savr TM GM tomatoes necrosis and stomach erosion were reported, whereas lectin induced proliferative growth is observed in their stomach due to GM potatoes expressing Galanthus nivalis (GNA). Glomerular stomach erosions can lead to severe hemorrhage in patients especially old aged on nonsteroidal anti-inflammatory agents (Pusztai et al., 2003). Doing research on gene transfer has resulted in the manufacture of the aquatic species with better abilities in areas such as resistance of disease, growth, tolerating cold, and metabolism of plant-based diets. Current advances include the development of successful research into boosting disease resistance and patented gene transfer methods. The majority of environmental and human health concerns take place despite of the potential for GMOs in aquaculture. Escapement of transgenic fish into the wild is one of the major problems, where they could disturb natural gene pools during reproduction with wild species, and the probable hazardous impacts of introducing transgenics into the aquatic and human food chains (Rasmussen & Morrissey, 2007). In one study it was reported that genetic manipulation process often led to toxicity. However, in humans or animals fed genetically manipulated corn, soya, and canola over an extended period of time (Pusztai et al., 2003). Constant inflammation and

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proliferative effect on the gastrointestinal tract take place due to few GM plants that may eventually lead to cancer after years. While there are only a small number of extended studies on GM foods that give information regarding its impact on the liver. Modification in functioning and cell structure of liver has been observed in mice fed with GM soya (Malatesta et al., 2002, 2003, 2005). After consumption of raw rice expressing GNA changes have been observed in hepatic enzymes (Poulsen et al., 2007). Hepatocellular damage can occur due to modifications in hepatocyte cells and enzymes. An increase in triglycerides level in females is observed due to consumption of Mon863 corn in rats (Seralini et al., 2007). Patented invention of a line of Atlantic salmon capable of better growth and feed conversion efficiency is responsible for a large amount of progress with transgenic GHs. This product has been handed to a large biotechnology business, and regulatory approval for commercial use in the United States and Canada is now pending. 9.2.9 EFFECTS OF GM FOOD ON PANCREAS After consuming GM soybean, a negative effect was seen in pancreatic acinar cells of mice, as well as a high synthetic rate of zymogen granules with minimal alpha-amylase (Malatesta et al., 2003). Other than liver harmful impact of GM crops has also been observed in kidney. In research conducted by DuPont, rats fed GM corn 1507 had smaller kidneys (MacKenzie et al., 2007), whereas utilization of Mon863 corn in rats led to lower urine sodium and phosphorus excretion in male rats. Tubular degenerative changes and increase in focal inflammation which are characteristics of a classic progressive nephropathy are observed due to consumption of GM foods (Seralini et al., 2007). In one study it was stated that those rats fed with GNA rice had shown raise in their creatinine plasma concentration either due to elevated quantity of water consumption or some sort of renal effect (Poulsen et al., 2007). Salmon fed GM soybeans have higher acid phosphatase activity and renal lysozyme levels (Bakke-McKellep et al., 2007). 9.2.10 MODIFICATIONS IN HEMATOLOGY OF ANIMALS Serum-facilitated allergen inhibition presentation is mediated by serum IgG. The presence of enhanced IgG Abs triggers the IgG response,

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indicating the existence of an allergic response (van Neerven et al., 1999). However, in one study it was reported that antigen specific IgG does not show a correlation to clinical allergy (Germolec et al., 2003). However, in male rats, GM corn Mon863 causes an increase in white blood cell counts (Seralini et al., 2007). A considerable amount of reduction in red blood cell count of females was reported in DuPont’s experiment in rats fed food containing GM corn 1507 (MacKenzie et al., 2007) while blood development with fewer immature red blood cells found in rats owing to eating of GM corn Mon863 and changes in blood chemistry (Seralini et al., 2007). Decrement in platelets, monocytes ratio in female rats and rise in the granulocyte ratio observed in male rats due to Bt with VIP insecticidal protein gene (Peng et al., 2007). In DuPont’s sub-chronic feeding trial, rats fed with GM corn 1507 showed a decrease in eosinophil content (MacKenzie et al., 2007). Swelling of the lymph nodes and reduced weight of the mesenteric was also reported in one study in which rats were given a diet containing GNA rice (Poulsen et al., 2007). 9.2.11 FOOD ALLERGENICITY TESTING TO PREVENT HEALTH ISSUES DUE TO INTAKE OF GE FOODS Allergenicity is an adverse effect of various foods, trees, chemicals, drugs, and cosmetics. Around 1% to 6% of the population has self-reported lifetime allergic responses to most common allergens which are used in day-to-day life, such as milk, peanut, soy, nuts, fish, etc. (Nwaru et al., 2014). To avoid any detrimental consequences on human and animal health, it is possible to investigate the potential allergenicity of a food or food product developed from a GE crop. Animal testing for checking allergens in food is not enough for allergy assessment. Research efforts are going to discover an animal product that predicts sensitivity to allergy. Furthermore, researchers have relied on a number of indirect methods to establish whether an allergic response is caused by a protein added to a meal using genetic engineering techniques or if the protein appears in the food as an unintentional result of genetic engineering. Allergic characteristics comprising endogenous protein concentrations must be evaluated since genetic engineering has the potential to increase their concentration. A description of how allergen testing is performed recommended by the Codex Alimentarius Commission (EFSA et al., 2010, 2011a) is

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presented in Figure 9.2. It shows EPA testing of the Bt toxin Cry1F, which is frequently used in this method. This technique is based on the idea that any protein gene from a plant that is known to cause food allergies has a higher chance of causing allergenicity than any other gene from a plant that does not cause an allergic reaction. If the introduced protein resembles a protein that is already known to be an allergen, it becomes suspect and should be tested in people who are allergic to the related protein. If a protein does not fall into any of the above mentioned categories and is not digested by stimulated gastric fluid, it may constitute a new food allergy.

FIGURE 9.2 A flow chart depicting the process for testing the allergenicity of a newly added protein in genetically modified (GM) organisms. Source: EFSA et al. (2010, 2011a).

9.3 CONCLUSION Based on an analysis of relevant research, facts demonstrate that employing rDNA technology may have a negative influence on the environment, human health, and animals, either directly or indirectly. The GMO deoxyribonucleic acid (DNA) that usually have antibiotic and pest resistant genes and the changed nutritional value may have an undesirable effect to

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human health. Ingestion of GMOs led to enhancement of the mortality rate among male rats. In addition to these, the consumption of GE foods may lead to the disturbance to human microbiome. Its undesirable impact in the human digestion resulting in digestive disorders can show the way to other health issues. Other accidental effect may include allergenicity, carcinogenicity, anti-nutritional effect, and toxicity. The development of GE crops can affect the lower form of life and can create trouble in the ecosystem. As a result, research have demonstrated that GM crops are hazardous to both the microorganism soil biota and invertebrate. Additionally, genetically modified (GM) food is not a natural food for humans; it is made by undergoing numerous genetic modification and alteration. Since it’s not a natural food, it is obvious to observe some unpredictable dangers to human health. However, it should be kept in mind the widespread controversy of GMOs to human health and to the lower form of life, taking the risk is not worthwhile for this time. It’s important to encourage organic farming and always choose organic foods for a better and improved health. KEYWORDS • • • • • • •

deoxyribonucleic acid European patent convention granulocyte colony-stimulating factors intellectual property rights organic foods severe acute respiratory syndrome coronavirus World Health Organization

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Haygood, R., Ives, A. R., & Andow, D. A., (2003). Consequences of recurrent gene flow from crops to wild relatives. Proceedings of the Royal Society of London: Series B: Biological Sciences, 270(1527), 1879–1886. Ho, M. W., Ryan, A., & Cummins, J., (2000). CaMV 35S promoter fragmentation hotspot confirmed, and it is active in animals. Microbial Ecology in Health and Disease, 12(3), 189. Hodgson, J., (2000). Scientists avert new GMO crisis. Nature Biotechnology, 18(1), 13–13. Hohlweg, U., & Doerfler, W., (2001). On the fate of plant or other foreign genes upon the uptake in food or after intramuscular injection in mice. Molecular Genetics and Genomics, 265(2), 225–233. James, C., (2005). Global Status of Commercialized Biotech/gm Crops (pp. 37–40). ISAAA Briefs No. 34, The International Service for the Acquisition of Agribiotech Applications, Ithaca, New York. Jennings, J. C., Albee, L. D., Kolwyck, D. C., Surber, J. B., Taylor, M. L., Hartnell, G. F., Lirette, R. P., & Glenn, M. K. C., (2003b). Attempts to detect transgenic and endogenous plant DNA and transgenic protein in muscle from broilers fed yield Gard corn borer corn. Poult. Sci., 82, 371–380. Jennings, J. C., Kolwyck, D. C., Kays, S. B., Whetsell, A. J., Surber, J. B., Cromwell, G. L., Lirette, R. P., & Glenn, K. C., (2003). Determining whether transgenic and endogenous plant DNA transgenic protein are detectable in muscle from swine fed roundup ready soybean meal. J. Anim. Sci., 81, 1447–1455. Kaynar, P., (2009). A general perspective on genetically modified organisms (GMOs). Turkish Journal of Hygiene and Experimental Biology, 66(4), 177–185. Kremer, R., Means, N., & Kim, S., (2005). Glyphosate affects soybean root exudation and rhizosphere micro-organisms. International Journal of Environmental Analytical Chemistry, 85(15), 1165–1174. Layton, R., Smith, J., Macdonald, P., Letchumanan, R., Keese, P., & Lema, M., (2015). Building better environmental risk assessments. Front. Bioeng. Biotechnol., 3, 110. Lu, B. R., & Yang, C., (2009). Gene flow from genetically modified rice to its wild relatives: Assessing potential ecological consequences. Biotechnol. Adv., 27, 1083–1091. MacKenzie, S. A., Lamb, I., Schmidt, J., Deege, L., Morrisey, M. J., Harper, M., Layton, R. J., et al., (2007). Thirteen week feeding study with transgenic maize grain containing event DAS-Ø15Ø7-1 in Sprague–Dawley rats. Food Chem. Toxicol., 45, 551–562. Magnusson, M. K., & Hursti, U. K. K., (2002). Consumer attitudes towards genetically modified foods. Appetite, 39, 9–24. Malatesta, M., Caporaloni, C., Gavaudan, S., Rocchi, M. B. L., Serafini, S., Tiberi, C., & Gazzanelli, G., (2002). Ultrastructural morphometrical and immunocytochemical analyses of hepatocyte nuclei from mice fed on genetically modified soybean. Cell Struct. Funct., 2, 173–180. Malatesta, M., Caporaloni, C., Rossi, L., Battistelli, S., Rocchi, M., & Tonucci, F., (2003). Ultrastructural analysis of pancreatic acinar cells from mice fed on genetically modified soybean. J. Anat., 201, 409–415. Malatesta, M., Tiberi, C., Baldelli, B., Battistelli, S., Manuali, E., & Biggiogera, M., (2005). Reversibility of hepatocyte nuclear modifications in mice fed on genetically modified soybean. Eur. J. Histochem., 49, 237–242.

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Martin, S. A. M., Vilhelmsson, O., M´edale, F., Watt, P., Kaushik, S., & Houlihan, D. F., (2003). Proteomic sensitivity to dietary manipulations in rainbow trout. Biochim. Biophys. Acta, 1651, 17–29. Mercer, D. K., Scott, K. P., Bruce-Johnson, W. A., Glover, L. A., & Flint, H. J., (1999). Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva. Appl. Environ. Microbiol., 65, 6–10. Mertens, M., (2008). Assessment of Environmental Impacts of Genetically Modified Plants BfN – Skripten 217. Federal Agency for Nature Conservation, New York, USA. Nordlee, J. A., Taylor, S. L., Townsend, J. A., Thomas, L. A., & Bush, R. K., (1996). Identification of a Brazil-nut allergen in transgenic soybeans. New England Journal of Medicine, 334(11), 688–692. Nwaru, B. I., Hickstein, L., Panesar, S. S., Roberts, G., Muraro, A., Sheikh, A., & EAACI Food Allergy and Anaphylaxis Guidelines Group, (2014). Prevalence of common food allergies in Europe: A systematic review and meta‐analysis. Allergy, 69(8), 992–1007. Ostaszewska, T., Dabrowski, K., Palacios, M. E., Olejniczak, M., & Wieczorek, M., (2005). Growth morphological changes in the digestive tract of rainbow trout (Oncorhynchus mykiss) pacu (Piaractus mesopotamicus) due to casein replacement with soybean proteins. Aquaculture, 245, 273–286. Paparini, A., & Romano-Spica, V., (2004). Public health issues related with the consumption of food obtained from genetically modified organisms. Biotechnology Annual Review, 10(1), 85–122. Peng, D., Chen, S., Ruan, L., Li, L., Yu, Z., & Sun, M., (2007). Safety assessment of transgenic Bacillus thuringiensis with VIP insecticidal protein gene by feeding studies. Food Chem. Toxicol. Phipps, R. H., Beever, D. E., & Humphries, D. J., (2002). Detection of transgenic DNA in milk from cows receiving herbicide tolerant (CP4EPSPS) soybean meal. Livestock Production Science, 74(3), 269–273. Phipps, R. H., Deaville, E. R., & Maddison, B. C., (2003). Detection of transgenic and endogenous plant DNA in rumen fluid, duodenal digesta, milk, blood, and feces of lactating dairy cows. Journal of Dairy Science, 86(12), 4070–4078. Poulsen, M., Kroghsbo, S., Schrøder, M., Wilcks, A., Jacobsen, H., Miller, A., Frenzel, T., et al., (2007). A 90-day safety study in Wistar rats fed genetically modified rice expressing snowdrop lectin Galanthus nivalis (GNA). Food Chem. Toxicol., 45, 350–363. Pray, C. E., Ramaswami, B., & Kelley, T., (2001). The impact of economic reforms on R&D by the Indian seed industry. Food Policy, 26(6), 587–598. Pusztai, A., Bardocz, S., & Ewen, S. W. B., (2003). Genetically modified foods: Potential human health effects. In: D’Mello, J. P. F., (ed.), Food Safety: Contaminants and Toxins (pp. 347–372). CAB International, Wallingford Oxon, UK. Rasmussen, R. S., & Morrissey, M. T., (2007). Biotechnology in aquaculture: Transgenics and polyploidy. Comprehensive Reviews in Food Science and Food Safety, 6, 2–16. Rauschen, S., Eckert, J., Schaarschmidt, F., Schuphan, I., & Gathmann, A., (2008). An evaluation of methods for assessing the impacts of Bt‐maize MON810 cultivation and pyrethroid insecticide use on Auchenorrhyncha (planthoppers and leafhoppers). Agricultural and Forest Entomology, 10(4), 331–339.

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Richards, H. A., Halfhill, M. D., Millwood, R. J., & Stewart, C. N., (2003). Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants. Plant Cell Reports, 22(2), 117–121. Saltik, A., (2010). Genetically modified food and public health. In: Aslan, D., & Şengelen, M., (eds.), Different Aspects of Genetically Modified Organisms. Physicians Room in Ankara (pp. 33–42). MatTek Press, Ankara. Samsel, A., & Seneff, S., (2013). Glyphosate’s suppression of cytochrome P450 enzymes and amino acid biosynthesis by the gut microbiome: Pathways to modern diseases. Entropy, 15(4), 1416–1463. Saxena, D., & Stotzky, G., (2000). Insecticidal toxin from Bacillus thuringiensis is released from roots of transgenic Bt corn in vitro and in situ. FEMS Microbiology Ecology, 33(1), 35–39. Saxena, D., Stewart, C. N., Altosaar, I., Shu, Q., & Stotzky, G., (2004). Larvicidal Cry proteins from Bacillus thuringiensis are released in root exudates of transgenic B. thuringiensis corn, potato, and rice but not of B. thuringiensis canola, cotton, and tobacco. Plant Physiology and Biochemistry, 42(5), 383–387. Schmidt-Dannert, C., Umeno, D., & Arnold, F. H., (2000). Molecular breeding of carotenoid biosynthesis pathways. Nat. Biotechnol., 18, 750–753. Schubbert, R., Renz, D., Schmitz, B., & Doerfler, W., (1997). Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA. Proc. Natl. Acad. Sci. USA, 94, 961–966. Seralini, G. E., Cellier, D., & De Venomois, J. S., (2007). New analysis of a rat feeding study with a genetically modified maize reveals signs of hepatorenal toxicity. Arch. Environ. Contam. Toxicol. [Epub ahead of print]. SPO (State Planning Organization), (2000). Report of Special Commission on Five-year Development Plan, Biotechnology and Biosecurity: Progress Project Proposal on National Molecular Biology, Modern Biotechnology and Biosecurity (p. 69). Ankara. Squire, G. R., Hawes, C., Bohan, D. A., Brooks, D. R., Champion, G. T., Firbank, L. G., & Young, M. W., (2005). Biodiversity Effects of the Management Associated with GM Cropping Systems in the UK. Defra, London. Szénási, A., Pálinkás, Z., Zalai, M., Schmitz, O. J., & Balog, A., (2014). Short-term effects of different genetically modified maize varieties on arthropod food web properties: An experimental field assessment. Scientific Reports, 4(1), 1–7. Tangley, L., (1983). Engineered Organisms in the Environment. Not yet, Biosei., B81-692. Tepfer, M., Gaubert, S., Leroux-Coyau, M., Prince, S., & Houdebine, L. M., (2004). Transient expression in mammalian cells of transgenes transcribed from the Cauliflower mosaic virus 35S promoter. Environmental Biosafety Research, 3(2), 91–97. Turkyilmaz, S., & Esendal, M. O., (2002). Polymerase chain reaction and its use in microbiology. KAFKAS University Journal of Veterinary Medicine, 8(1), 71–76. Van, N. R. J. J., Wikborg, T., Lund, G., Jacobsen, B., Brinch-Nielsen, A., & Arnved, J., (1999). Blocking antibodies induced by specific allergy vaccination prevent the activation of CD4+ T cells by inhibiting serum-IgE-facilitated allergen presentation. J. Immunol., 163, 2944–2952. Weil, J. H., (2005). Are genetically modified plants useful and safe? IUBMB Life, 57, 311–314.

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Yu, H. L., Li, Y. H., & Wu, K. M., (2011). Risk assessment and ecological effects of transgenic Bacillus thuringiensis crops on non-target organisms. J. Integr. Plant Biol., 53, 520–538.

CHAPTER 10

Genetic Engineering and Agricultural Sciences MUHAMMAD ISHTIAQ, MUHAMMAD WAQAS MAZHAR, and MEHWISH MAQBOOL

Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur – 10250, Azad Jammu and Kashmir (AJK), Pakistan

ABSTRACT Selective breeding and artificial selection find history some 10,000 years ago in Southwest Asia, and since then, genetic modifications through plant breeding techniques has emerged as a fascinating field to improve the crop and agriculture however, the plant breeding techniques could not evolve well enough to meet growing food demands for escalating human population. In modern times, agricultural biotechnology is offering tremendous scope and potential to provide food security through crop improvement techniques and protocols following principles of genetic engineering. Modern genetic engineering techniques have promised improved shelf life, better agronomic vigor, and pest-free traits to the field crops, and through these interventions, desirable traits have been induced in the crops. In this chapter, a comprehensive account on the use of genetic engineering in agriculture sciences has been presented.

Genetic Engineering, Volume 2: Applications, Bioethics, and Biosafety. Tariq Ahmad Bhat & Jameel M. Al-Khayri (Eds.) © 2023 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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10.1 GENERAL BACKGROUND OF GENETICALLY MODIFIED (GM) CROPS GM crops are produced by introducing characterized foreign genes in a crop through the recombinant DNA technology (RDT). These characterized genes are often termed as “transgenes” and the plant having these transgenes are termed as transgenic plants or genetically modified (GM) plant. The GM crops are inevitable in modern times as our earth is facing the climate change and escalating human population scenario. Although the genetic modifications can be achieved through the selective breeding and artificial selection methods through mutagenesis and intergeneric hybridization however, these transformations are not biorational as several uncharacterized genes are transferred. The genetic engineering offers better technology and better method for gene manipulation compared to conventional breeding programs. Although the debate persists among opponents and supporters of the GM crops, yet the area covered by the GM crops is escalating on the globe due to their better agronomic performance (Table 10.1) and in 2015 the area under cultivation with GM crops was 179 million ha. Planting GM crops has increased yields by 22% providing the farmers a 68% more profit comparatively (Kumar et al., 2020). The Flavr Savr tomato were the first commercially grown crop in 1994 and since then a number of cereals and other cash crops are being planted including rice, soybean, cotton, sugarcane, corn, sugar beet and rapeseed. TABLE 10.1 Top 10 GM Crops Growing Countries Worldwide in 2019 (Area Wise in Million Hectares) Rank No. Name of Countries 1. United States of America 2. Canada 3. China 4. Brazil 5. Argentina 6. South Africa 7. India 8. Paraguay 9. Bolivia 10. Pakistan

Area 71.5 12.5 3.2 52.8 24 2.7 11.9 4.1 1.4 2.5

Genetically Modified Crop Names Maize, soybean, cotton, canola, sugar beet, alfalfa, papaya, squash Canola, maize, soybean, sugar beet Cotton, papaya, poplar, tomato, sweet pepper Soybean, maize, cotton Soybean, maize, cotton Maize, soybean, cotton Cotton Soybean, maize, cotton Soybean Cotton

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A total of 525 transgenic interventions in 32 crops have been authorized for cultivation. To overcome the unknown fears and known concerns with the application of foreign genes into the crop plants cisgenesis and intragenesis were introduced. Both of these techniques allow the safe manipulation of transgenes through closely related and sexually compatible gene pool. Furthermore, the advancement in genetic engineering has led to development of genome editing site-specific nucleases (SSNs), viz., zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system overcoming the fears of mutagenesis associated with genetic engineering. Genetic engineering through recombinant DNA (rDNA) (Figure 10.1) involves following fundamental protocols: i. Generation of DNA fragment with characterized gene sequence through restriction endonucleases; ii. Separating the DNA fragments using gel-electrophoresis; iii. Sorting the specific sequence through nucleic acid hybridization techniques; iv. Manipulation of DNA in the cell; v. Amplification of DNA using polymerase chain reaction (PCR) and gene cloning protocols.

FIGURE 10.1

Basic steps in recombinant DNA technology.

Source: Rout et al. (2018).

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10.2 APPLICATIONS OF GENETIC ENGINEERING PRINCIPLES (GEPS) IN AGRICULTURAL SCIENCE 10.2.1 BETTER AGRONOMIC TRAITS The application of GEPs revolutionized agricultural sciences. Planting of pathogen resistant and herbicides tolerant GM crops led to 5–10% increase in the yield in the year 1996–1997 (James et al., 1998). In developing countries warming at poles and coastal flooding have promoted yield losses. Furthermore, the changing climates are bringing about more pest encounters and invasive species. In tropics and subtropics the post-harvest yield losses are also very high as the climate of these areas promotes mycotic diseases. The farmers are smallholders, and most of them are not aware of the modern approaches towards post-harvest operations due to which around 15% of yields are lost every year. Modern plant genetic engineering allows to solve many of these problems. The GEPs enable to produce disease and pest infestation resistant varieties. The cultivation of insect resistant varieties of cotton and maize produced using Bacillus thuringiensis (Bt) endotoxin is an example. Furthermore, genetic engineering has enabled the crop scientists to develop the delayed ripening varieties of tomatoes. This application of genetic engineering might be potentially helpful for tropical fruit tree species which ripen fast and due to poor storage and transportation facilities, a great number of yields is lost before reaching the ultimate consumer. 10.2.2 ABIOTIC STRESS MANAGEMENT Climate change is inducing severe droughts, heat storms, frost, heat stress and saline environments in the vicinity of agricultural lands across the globe. These abiotic stresses are major yield limiting forces faced by the plants. Modern plant genetic engineering is trying to answer these biological problems. The tomato plants are being engineered genetically against water stress with a DNA cassette containing an Arabidopsis C repeat/ dehydration-responsive element binding factor 1 (CBF1) complementary DNA (cDNA) and a NOS terminator, driven by a cauliflower mosaic virus 35S (CaMV35S) promoter (Tsai-Hung et al., 2002). The protein called osmotin plays important role to combat abiotic stress. The expression of tobacco osmotic gene induced salt tolerance in transgenic chili plants

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(Subramanyam et al., 2011). Similarly, the expression of SAMDC gene from yeast in tomato resulted in induction of tolerance mechanism in plants against heat stress (Cheng et al., 2009). 10.2.3 BIOTIC STRESS MANAGEMENT

Around 10% to 16% of the global agricultural yields are lost every year due to pathogen and crop infestations (Chakraborty and Newton, 2011). The application of genetic engineering is enabling the biotic stress management by engineering plants with pest resistant traits that is not possible through conventional cross breeding techniques provided the resistance to biotic stress genes do not exist in the naturally occurring gene pool. The application of pesticides is not an eco-friendly practice since the pesticides may enter in the food chains, and these pesticides eventually yield plant varieties with chemical resistance and increased pesticides tolerance in insects can be developed (Wahab, 2009). Currently 71.5 million hectares area is under cultivation with genetically engineered pest resistant cultivars in USA. The cultivation of 32 genetically amended crops have served a 68% increase in farmer profit globally. 10.2.3.1 PEST RESISTANCE The Bt gene of Bacillus thuringiensis is being exploited globally to develop pest resistant plant varieties. Fischhoff et al. (1987) reported the pest resistance Bt tomato plants which showed resistance against pests Spodoptera litura and Heliothis virescens. Similarly, the potato verities resistant against Colorado potato beetle have been genetically engineered (Shelton et al., 2002). Furthermore, the genetically engineered Brassica oleraceae var. capitata has been raised (Paul et al., 2005) which is resistant against P. xylostella. 10.2.3.2 DISEASE RESISTANCE More than 50% of food is lost worldwide due to fungal attacks and diseases caused by microbes. The orthodox approaches of plant breeding

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involving conventional selective breeding techniques are useless in developing disease resistant varieties of the plants due to the non-availability of disease resistance genes in the naturally occurring genome. However, genetic engineering is useful in developing disease resistant varieties of the plants, especially commercially important cereal crops. Induction of tolerance against viral diseases in fruit crops have been achieved by manipulating the CP gene of the virus against diseases such as grape fan leaf virus and PPV. Similarly, PRSV disease resistant papaya is being cultivated in the USA. Praveen et al. (2010) developed transgenic plants of tomato with an AC4 gene-RNAi construct, and the transgenic plants were found to show the suppression of tomato leaf curl virus activity. 10.2.3.3 HERBICIDE RESISTANCE Using the principles of genetic engineering, higher plants are being engineered to tolerate herbicides. For instance, the herbicide glyphosate works as an inhibitor of enzyme 5 enolpyruvylshikimate-3-phosphate synthase (EPSP) in herbs in higher plants, so there was a need to engineer plants against this inhibitor. Shah et al. (1986) developed a recombinant EPSP synthase gene using CaMV and produced its high expression in petunia cells which resulted in petunia plants tolerant to glyphosate. Similarly, the plants tolerant to Basta herbicide were reported by Sriporaya et al. (2006). 10.2.4 INCREASED FORTIFICATION Early versions of GM crops were developed mainly to equip the plants against biotic and abiotic stressors however the focus now has shifted towards genetic modification to achieve higher dietary values from the food products. Malnutrition is a serious problem in developing countries so currently, the major focus of the genetic engineering is to develop nutritionally better versions of transgenic crops to overcome malnourishment among people worldwide. 10.2.5 IMPROVED SHELF-LIFE AND DELAYED RIPENING Improper storage conditions leave the fruits and vegetables to rot and decay faster soon after harvest. Due to this issue, farmers experience postharvest yield losses and hence suffer economically. One of the aims of

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the genetic engineering was to delay the ripening of fruits and improve the storage life of fruits to minimize the post-harvest yield losses (Flavr Savr tomato). Furthermore, the improved vitamin and dietary contents of fruit was another aim of developing a GM crop. Using the principles of genetic engineering, the fruits have been modified for delayed ripening by modifying the activity of polygalacturonase enzyme which is involved in fruit ripening process by loosening cell wall. Similarly, the ethylene production is also being monitored to delay fruit ripening process. The Flavr Savr tomatoes were produced and cultivated for this purpose. Later on, other tomato varieties with increased shelf-life were developed through antisense RNA inhibition of 1-aminocyclopropane-1-carboxylate (ACC) synthase or ACC oxidase and two ethylene precursors (Figure 10.2).

FIGURE 10.2

Commercialization goals of genetically modified plants in the 21st century.

10.3 GM CROPS: AREAS OF CONCERN Although the transgenic versions of crops have served economic gains and potential benefits to the farmers in terms of better input and output traits, however there are some areas of concern associated with these crops (Kumar et al., 2020).

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10.4 BIOHAZARDS OF GM CROPS The introduction of GM crops in natural environment brings some biosafety issues related to environmental integrity and ecosystem sustainability (Suzei et al., 2009). GM crops bring risk of inducing allergic reactions and symptoms of potential health issues. The example of StarLink maize (a transgenic version of maize approved for animal feed in USA since 1998) has not been approved for human use since it brings some allergic reactions and symptomatology usually associated with allergens. The Seralini affair (Seralini et al., 2012) is perhaps best explained biosafety issue raised due to intake of transgenic NK-603 maize. In rats fed with this GM crop suffered chronic kidney disease, hepatic congestion, necrosis, and female mortality. Seralini et al. (2014) again published the work to fuel the discussion on GM crops and biosafety issues related to their application. The transfer of antibiotic resistance genes from GM products to the digestive tract of the human and other animals presents another area of concern. Such antibiotic resistance genes may transform gut microbes to be antibiotic resistant (Heritage, 2004). The environmental risks associated with the introduction of GM crops is that the pollen produced by a particular GM crop might travel to conventional food crops cultivars (Yan et al., 2015). This will lead to the development of herbicide resistant weeds popularly known as superweeds. The incidence of glyphosate resistant weeds is being reported on the more common basis from the cropping system with GM cultivars (Heap, 2014). 10.5 DEVELOPMENT OF RESISTANCE The planting of GM crops may induce resistance in the targeted weeds and insects owning to elevated selection pressures. That elevated selection pressures might lead to the origin of new species and biotypes. The gene flow in the form of pollens to the wild relatives of the crops may lead to the development of superweeds, and all of such instances might be alarming for ecosystem integrity and biosafety (Gilbert., 2013). For instance, field evolved pest resistance to Bt maize has been reported for three major pest species, viz., Busseola Fusca (African stem borer) in South Africa to cry1Ab expressing corn (Rensburg 2007), Spodoptera frugiperda (fall armyworm) in Puerto Rico to cry1F expressing corn (Storer et al., 2010) and D. virgifera virgifera (western corn rootworm) in USA to cry3Bb expressing corn (Gassmann et al., 2011).

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10.6 HIGH PRICES FOR GM CROP SEEDS, COMMERCIALIZATION COSTS, AND PATENTING ISSUES The seeds and planting material of a GM crop is patentable and thus multinational companies such as Monsanto and Bayer Crop Science set high prices for the patent seeds. Furthermore, the oligopoly of these companies renders small firms and markets deployed against the rights of buying and patenting. Thus, the smallholder farmer suffers a great deal as the seeds due to patenting issues are not reachable, and prices are very high for these varieties. For better results better commercialization protocols are required to be followed. The high commercialization cost and lengthy protocols adopted in crop commercialization and adoption are another hurdle raising further concerns in GM crop sector. The companies involved in the commercialization of the transgenic varieties show little interest due to the lengthy approval process involved in the adoption of GM crops (Kumar et al., 2020). 10.7 MONARCH BUTTERFLY CONTROVERSY: EVIDENCE OF GM INTERFERENCE WITH NON-TARGETS Losey et al. (1999) reported that Danaus plexippus commonly known as monarch butterfly is an indirect target of GM Bt maize. The controversy originated as Losey, and his colleagues experimented with the larvae of the monarch butterflies to feed on milkweed leaves which were dusted with pollens from GM Bt maize. The larvae were adversely affected, and their mortality rate was found higher due to GM Bt maize pollens. However, the later studies suggested by Bower et al. (2012) reported that herbicide glyphosate is the main reason behind the decline in the butterfly population as the herbicide kills the feed of monarch butterfly, i.e., milkweed. However, the study put forward concerns regarding the biosafety issues related to practicing transgenic crops to non-targeted organisms. 10.8 NEW TRENDS IN GENETIC MODIFICATIONS 10.8.1 GENOME EDITING TECHNOLOGIES The advent of modern interventions in the genetic engineering and biotechnology has led to discovery of novel protocols and set of methodologies

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which might be successful in editing the certain genes to entire genome. These techniques are based on the use of certain nucleases which can edit a gene. Furthermore, the synthetic oligonucleotides provide a clear-cut basis for creating a point mutation in the DNA molecule to be edited (Songstad et al., 2017). The use of genome editing technologies such as ZFNs and TALENs might be fruitful in genome editing both operating through the same underlying mechanism of fusing two protein domains involving Fok1 endonuclease. A double strand break to a specific target site on the DNA molecule is produced by a pair of ZFN or TALEN thereby activating the endogenous DNA biological repair system (Kim et al., 1996). Theoretically, the target site can be of any length, however the context dependent assembly of ZFN modules limits the target site size (Sander et al., 2011). Unlike zinc fingers (ZF), each TAL effector (TALE) domain binds a single nucleotide specifically (Christian et al., 2010). The target sites in the genome can be chosen by customizing ZFNs (or TALENs) design via selecting appropriate combinations of different ZF (or TALE) domains, respectively. ZFN- or TALEN-mediated gene editing techniques have some disadvantages, such as cumbersome protein engineering steps, high costs and are difficult to multiplex. Among the genome editing tools, CRISPR/Cas9 is gaining popularity due to its wide range of applications in the treatment of human diseases and genetic engineering. This editing tool consists of two components which include single stranded RNA and enzyme Cas9 endonuclease. This single stranded RNA molecule binds to the targeted sequence on the DNA molecule through the double strand breaks created by Cas9 endonuclease. This whole double strand break followed by RNA base pairing at the target site activates the innate DNA repair mechanism. The RNA guided CRISPR genome editing tool is more appropriate for genome editing of crops. Furthermore, the convenience in application of the Cas9 system as compared to other genome editing tools such as TALEN and ZFN proves it best gene editing tools (Figure 10.3). 10.8.2 CISGENESIS AND INTRAGENESIS Cisgenesis is a genetic modification protocol which involves the insertion of a natural gene sequence along with the native intron and promoterterminator sequence in the sense orientation. Usually, the natural gene

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FIGURE 10.3 Modern genome editing tools used in crop genetic engineering. (A) Zinc finger editing using Fok 1; (B) TALEN using Fok 1; and (C) CRISPR using Cas9 endonuclease to create breaks in targeted site followed by endogenous repair mechanism. Source: Sanagala et al. (2017).

sequence is taken from the crop itself or the sexually compatible naturally occurring gene pool (Schouten et al., 2006). Unlike the orthodox approaches to plant breeding the cisgene being manipulated contains only the characterized gene sequence and uncharacterized gene elements are not the part of gene manipulation. Through the cisgenesis approach crop scientists are now able to induct novel traits in the plants, especially the disease resistance. The diseases such as late blight of potato is now genetically curable using the principles of genetic engineering as reported by Haverkort et al. (2009). Vanblaere et al. (2011) reported the extenuation of the apple scab using the principles of cisgenesis similarly the work reported by Holme et al. (2012) reports that certain anti-nutrients such as phytate can be detoxified in barley. However, there has been no evidence yet in commercialization of a cisgenic event. On contrary to cisgenesis the intragenesis involves the manipulation the gene from sexually compatible genome but the promoter and terminator sequences are usually taken from certain other genes. All of such practices promote novelty in genetic recombinant products.

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10.9 CONCLUSION GM crops have the potential to provide food security to the inhabitants of this greenhouse planet which is already facing the risk of climate change and global warming. GM crops can serve humanity as these promise for better yield attributes and improved shelf life. However, the governments are aware of risks associated with the GM products, and there are challenges for the government for safe testing of GM products. A large proportion of the crop scientists, breeders, and the general public feel that the genetic engineering is inevitable, and it should be the part and parcel of human life as it is not affordable to ignore the advent of such a fascinating technology. Yet to move cautiously is the need of the hour since ecosystem integrity, environmental ethics and safe labeling of the GM product is the first priority which in not be compromised either. Authors recommend the better policy formulation in the manipulation of GM crops. KEYWORDS • • • • • •

Bacillus thuringiensis cisgenesis farm-scale evaluations Galanthus nivalis genetic diversity polymerase chain reaction

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Rommens, C. M., Haring, M. A., Swords, K., & Davies, H. V., (2007). The intragenic approach as a new extension to traditional plant breeding. Trends Plant Sci., 12, 397–403. Rout, G. R., & Peter, K. V., (2018). Genetic Engineering of Horticultural Crops. Academic Press. Sanagala, R., Moola, A. K., & Diana, R. K. B., (2017). A review on advanced methods in plant gene targeting. Journal of Genetic Engineering and Biotechnology, 15(2), 317–321. Sander, J. D., Dahlborg, E. J., Goodwin, M. J., Cade, L., Zhang, F., Cifuentes, D., Curtin, S. J., Blackburn, J. S., Thibodeau-Beganny, S., Qi, Y., et al., (2011). Selection-free zincfinger-nuclease engineering by context-dependent assembly (CoDA). Nat. Methods, 8, 67–69. Schouten, H. J., Krens, F. A., & Jacobsen, E., (2006). Cisgenic plants are similar to traditionally bred plants. EMBO Rep., 7, 750–753. Seralini, G. E., Clair, E., Mesnage, R., Gress, S., Defarge, N., Malatesta, M., Hennequin, D., & De Vendômois, J. S., (2014). Republished study: Long-term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize. Environ Sci Eur., 26, 14. Seralini, G. E., Clair, E., Mesnage, R., Gress, S., Defarge, N., Malatesta, M., Hennequin, D., & De Vendômois, J. S., (2012). Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize. Food Chem. Toxicol., 50, 4221–4231. Shah, D. M., Horsch, R. B., Klee, H. J., Kishore, G. M., Winter, J. A., & Tumer, N. E., (1986). Engineering herbicide tolerance in transgenic plants. Science, 233(4762), 478–481. Shelton, A. M., Zhao, J. Z., & Roush, R. T., (2002). Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Annu. Rev. Entomol., 47, 845–881. Songstad, D. D., Petolino, J. F., Voytas, D. F., & Reichert, N. A., (2017). Genome editing of plants. Crit. Rev. Plant Sci., 36, 1–23. Sripaoraya, S., Keawsompong, S., Insupa, P., Power, J. B., Davey, M. R., & Srinives, P., (2006). Genetically manipulated pineapple: Transgene stability, gene expression and herbicide tolerance under field conditions. Plant Breed., 125, 411–413. Storer, N. P., Babcock, J. M., Schlenz, M., Meade, T., Thompson, G. D., Bing, J. W., & Huckaba, R. M., (2010). Discovery and characterization of field resistance to Bt maize: Spodoptera frugiperda (Lepidoptera: Noctuidae) in Puerto Rico. J. Econ. Entomol., 103, 1031–1038. Subramanyam, K., Sailaja, K. V., Subramanyam, K., Rao, D. M., & Lakshmidevi, K., (2011). Ectopic expression of an osmotin gene leads to enhanced salt tolerance in transgenic chilli pepper (Capsicum annum L.). Plant Cell Tissue Organ Cult., 105, 181–192. Suzie, K., Ma, J. K. C., & Drake, P. M. W., (2008). Genetically modified plants and human health. J. R. Soc. Med., 101, 290–298. Tsai-Hung, H., Jent-turn, L., Yee-yung, C., & Ming-Tsair, C., (2002). Heterology expression of the Arabidopsis C repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato. Plant Physiol., 130, 618–626. Vanblaere, T., Szankowski, I., Schaart, J., Schouten, H., Flachowsky, H., Broggini, G. A., & Gessler, C., (2011). The development of a Cisgenic apple plant. J. Biotechnol., 154, 304–311.

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Wahab, S., (2009). Biotechnological approach in the management of plant pests, diseases and weeds for sustainable agriculture. J. Biopestic., 2, 115–134. Yan, S., Zhu, J., Zhu, W., Li, Z., Shelton, A. M., Luo, J., Cui, J., Zhang, Q., & Liu, X., (2015). Pollen-mediated gene flow from transgenic cotton under greenhouse conditions is dependent on different pollinators. Sci. Rep., 5, 15917.

CHAPTER 11

Construction of Recombinant DNA MOHAMMAD AMIN LONE and ANZAR A. SHAH

Department of Zoology, Government Degree College (GDC), Uri, Jammu and Kashmir, India

ABSTRACT Genetic engineering, also known as gene modification or gene editing, is a field of biotechnology that involves the direct manipulation of an organism's genetic material in order to modify or add traits. This can be done through a variety of techniques, such as the insertion of genetically modified DNA into an organism, or the disruption or suppression of certain genes. Genetic engineering has the potential to revolutionize the way we produce food, create new medicines, and address environmental challenges. However, it is also a controversial field, with many ethical, social, and environmental considerations. One of the key concepts in genetic engineering is the use of genetically modified organisms (GMOs). These are organisms whose genetic material has been modified using genetic engineering techniques. The goal of using GMOs is often to introduce new traits or characteristics into the organism, such as increased resistance to pests or diseases, or enhanced nutritional value. However, there are concerns about the safety of GMOs, both for the environment and for human consumption. Another important concept in genetic engineering is gene editing, which involves the precise modification of an organism's genetic material at the DNA level. Gene editing has the potential to be used for a wide

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range of applications, including the treatment of genetic diseases and the production of new medicines. However, there are also concerns about the potential ethical implications of gene editing, such as the creation of designer babies or the enhancement of human traits. Overall, genetic engineering is a complex and rapidly-evolving field with the potential to significantly impact many aspects of our lives. While it offers many benefits, it also raises important ethical, social, and environmental considerations that need to be carefully considered as the field continues to develop. 11.1 INTRODUCTION The enormous potential of recombinant DNA technology (RDT) has not been concealed from Stanley Cohen, Herbert Boyer, or their colleagues. At that time, it was suggested by Cohen, “It is possible to introduce genes into E. coli specific for metabolic or synthetic functions, such as photosynthesis or the production of antibiotics of other biological classes.” In 1973, a method has been developed to transfer genetic information (genes) from one organism to another. In their experiment, they could recombine two plasmids and clone the novel plasmid in E. coli. It gave the real kick-start to modern RDT and also laid the foundation for molecular biotechnology today. This approach, also termed as RDT, has allowed researchers to segregate specific genes, and insert them into host organisms. Human insulin used in the treatment of diabetes was the first marketed drug synthesized by RDT. The DNA sequence encoding human insulin was synthesized and transplanted into a plasmid that could be maintained in a common E. coli bacterium. Bacterial host cells were used as “bio-factories” to produce two peptide chains of human insulin. The insulin chains so formed are subjected to isolation followed by purification, and after purification, these chains are joined together to form full-fledged human insulin. This insulin can be used to treat diabetics who are allergic to the commercially available porcine (pig) insulin. RDT has benefitted many fields of research. However, its influence on biotechnology is extraordinary. RDT has provided a fast, effective, and potent way to create microorganisms with specific genetic attributes. Apart from micro-organisms, RDT tools have also enabled plants and animals to be genetically modified (GM). The advent of RDT has welcomed a new chapter for the creation of a wide range of therapeutic agents in sufficient quantities for the wellbeing

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of humans. These recombinant DNA (rDNA) products have revolutionized the treatment of certain diseases such as diabetes, asthma, atherosclerosis, heart attacks, and hemophilia and do not produce any allergic reactions (Hammer, McPhee, & Education, 2014). 11.2 RECOMBINANT DNA (RDNA)

This is an umbrella term that includes a set of experimental protocols that lead to the transfer of genetic information (DNA) from one organism to another. Encyclopedia of Britannica has defined recombinant DNA as “DNA molecules from two different species inserted into a host organism to produce new genetic combinations valuable for science, medicine, agriculture, and industry” [Hohn & Murray, 1977; https://www.britannica.com/ question/What-is-recombinant-DNA-technology. Accessed 12 January 2023]. The technology is also called gene cloning or molecular cloning. The following comprehensive format is frequently used in a DNA recombination experiment. The DNA (gene of interest) is mined from a donor organism, cleaved by enzymes, and linked to another DNA entity (a cloning vector) to form a new, rDNA molecule. The resulting DNA molecule is also termed as DNA construct. This DNA construct is transferred into and maintained within the host cell. The insertion of DNA into a bacterial host cell is referred to as transformation. These host cells that take up the DNA construct are known as transformed cells. Those transformed cells are identified and selected from the untransformed cells. 11.3 TOOLS OF THE TRADE In the late 1960s, the molecular biologists were frustrated as the research had evolved to the point where technical constraints hindered the progress. By the late 1960s, the most key requirements for the progression of genetic technology had been met. The genetic code could not be extended to incorporate a more comprehensive analysis of the gene. However, most of the advances have provided the impetus needed to manipulate genes to make them a reality. DNA ligase is a joining enzyme. It joins two strands of DNA together, which is a prerequisite for the construction of recombinant molecules. This was isolated in 1967. It is also regarded as a

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sort of molecular glue. That first restriction enzyme was isolated in 1970. The isolation of restriction enzyme was a significant step in the development of genetic engineering. Restriction enzymes (REs) are an important molecular scissors that cut DNA at precisely defined sequences. These enzymes can be used to produce DNA fragments suitable for association with other fragments. Thus, by 1970, the basic tools that were needed to build rDNA were now available. An engineer is someone who designs structures (for example, bridges, canals, and railways) and works according to an established plan. The term genetic engineering may be appropriate for someone involved in genetic manipulations. The toolbox or molecular tools of genetic engineering, which are the enzymes most commonly used in rDNA experiments, are briefly described: 1. Nucleases: A group of enzymes involved in nucleic acid degradation is called nucleases. These are of two types: i. DNAse: These are the enzymes, which hydrolyze DNA by breaking their phosphodiester bonds. ii. RNAse: These are the enzymes that break down the RNA molecules by hydrolyzing their sugar-phosphate backbone. These belong to two major categories: a. Exonuclease: These enzymes attack the nucleic acids at their terminal site only. They operate either on the 3’ or 5’ end of the linkage. b. Endonucleases: Those enzymes which can cut the phosphodiester bonds within a polynucleotide chain are termed as Endonucleases. There are some endonucleases, such as deoxyribonuclease I, which non-specifically, i.e., irrespective of any sequence, cut the DNA, while as, there are some enzymes, typically known as REs or restriction endonucleases, which cut the DNA only at a specific nucleotide sequence. 11.4 RESTRICTION ENDONUCLEASES – DNA CUTTING ENZYMES REs is a special group of nucleases that make specific cuts in DNA strands to generate fragments. These are referred to as chemical knives in genetic engineering. The enzymes are present in bacterial cells, such

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as E. coli, Bacillus haemophilus, Streptococcus, Thermus aquaticus, etc. In these organisms, these enzymes form part of a protection system called the restriction-modification system. They use these enzymes as chemical weapons against invading viruses or any other alien DNA. Molecular biologists, namely Werner Arber, Hamilton O Smith and Daniel Nathans discovered and characterized REs in the late 1960s and early 1970s (Britannica, n.d.) The restriction endonucleases were first discovered in E. coli. These enzymes have the potential to restrict the replication of bacteriophages by cutting viral DNA. Therefore, the addition of the methyl group to the host E. coli DNA protects it against cleavage. For this reason, the enzymes capable of restricting the viral replication are known as REs or restriction endonucleases. In this system, the restriction enzyme hydrolyzes all exogenous DNA seen in the cell. REs is of three types, namely: type I, type II, and type III. Type I and Type III enzymes detect specific nucleotide sequences in the DNA duplex, but they cut the nucleotide strand at a long distance away from the site of recognition. They are therefore not qualified when it comes to genetic engineering. The most widely used enzymes today are type II enzymes, which have a simple mechanism of action. Type II enzymes recognize a specific nucleotide sequence and cleave it in the vicinity of the recognition sequence. Type II enzymes are basically nucleases. Since they cut at an internal position in a DNA strand, they are referred to as endonucleases. Therefore, such enzymes may properly be classified as type II restriction endonucleases, although they are often simply called restriction enzymes (REs). In fact, they are the molecular scissors because they cute DNA at or near specific recognition sequences known as restriction sites. 11.4.1 NOMENCLATURE Restriction endonucleases are named after the bacterium from which they are isolated. The REs is named using the first letter of the genus and the first two letters of the species with strain observation and numerical letter for strain containing more than one enzyme (Nicholl, 2008). Therefore, an enzyme of a strain of Escherichia coli is called EcoRI, one of Bacillus amyloliquefaciens is BamHI, one from Haemophilus aegyptius is Hae III and so on. Further descriptors may be added, depending on the bacterial

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strain involved and, on the presence, or absence of extrachromosomal elements. Roman numeral denotes the order of discovery (Nicholl, 2008; Roberts & Murray, 1976). A couple of examples are given below: • EcoRI is from Escherichia coli: E = Escherichia; co = coli; R = strain; and I = 1st. • Hind III is from Haemophilus influenza: H = Haemophilus; in = influenza; d = strain; and the third endonucleases (III) to be discovered. • Sau3A is isolated from Staphylococcus (S); aureas (au); strain 3A. 11.4.2 RECOGNITION SEQUENCES The sequence recognized by the restriction enzyme to cut the DNA is called Restriction site or recognition site. Recognition site is predominantly GC rich. It consists of 4–8 base pairs. The ability of an enzyme to recognize a particular sequence is called sequence specificity. These short symmetric sequences of bases are termed Palindromic sequences. In Palindromic sequences, the base composition remains the same in both strands of DNA but in the opposite direction, i.e., sequences of bases are the same when read in 5’-3’ direction on both DNA strands. It is similar to palindromic word which reads the same in both the directions, e.g., MADAM, MALAYALAM (Figure 11.1).

FIGURE 11.1 A palindromic sequence.

Restriction endonucleases have a unique feature of attaching between the same two bases on the opposite strands, thereby causing a break in the two. These are categorized into two groups. One group of endonucleases produce short single stranded end known as sticky end which can join single stranded end of other DNA fragment

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having complementary sequence. Such ends are also known as staggered/ cohesive ends. These sticky/cohesive ends are produced by the REs because they cut the strands of DNA slightly away from the center of restriction site but between the same two bases present on the two strands. E-coRI (from E. coli) and BamHI (from Bacillus amyloliquifaciens) belong to this category (Figure 11.2).

FIGURE 11.2

Restriction enzymes produce DNA fragments with sticky ends.

If the DNA from different sources is cut by the same restriction endonucleases, they would produce complementary sticky ends. By using DNA ligase, the complimentary ends from the two DNAs can be joined end to end to generate a r DNA. The second group of endonuclease cuts both the strands of DNA at the same place so that single stranded pieces are not left on the ends. These ends without single stranded sequences are known as blunt ends. SmaI (from Serratia marcescens) and Sca I (from…) belong to this category (Figure 11.3).

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FIGURE 11.3

Restriction enzymes producing blunt ends.

Fragments with blunt ends may be used as such. Small linkers are sometimes added to the ends for separation of gene later on. Blunt ends may also be modified to cohesive ends by adding Poly A and Poly T sequences with the help of enzyme terminal transferase. Some of the most frequently used REs with their recognition sequences and the source of the enzyme strain from which they are obtained are listed in Table 11.1. TABLE 11.1 Recognition Sequences and Cutting Sites for Some Restriction Endonucleases Enzyme Recognition Sequence Cutting Sites BamHI 5’---GGATCC---3’

EcoRI

3’---CCTAGG---5’

Source of the Enzyme Bacillus amyloliquefaciens H

5’---G AATTC---3’

Escherichia coli

3’---CTTAAG---5’ HaeIII

5’----GGCC----3’

Haemophilus Aegyptus

3’----CCGG----5’ HpaI

5’---GTTAAC---3’ 3’---CAATTG---5’

Haemophilus parainfluenza

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TABLE 11.1 (Continued) Enzyme Recognition Sequence Cutting Sites

Source of the Enzyme

PstI

Providencia stuarii

5’---CTGCAG---3’ 3’---GACGTC---3’

Sau3A

5’---GATC----5’

Staphylococcus aureus

3’---CTAG----3’ SmaI

5’---CCCGGG---3’

Serratia maccescens

3’---GGGCCC---3’ SstI

5’---GAGCTC---3’

Streptomyces Stanford

3’---CTCGAG---5’

XmaI

5’---CCCGGG---3’ 3’---GGGCCC---5’

Xanthomonas malvacearum

11.4.3 USES OF RESTRICTION ENZYMES (RES) Restriction enzymes play very significant roles in biotechnology: • Restriction enzymes are used to cut a source DNA into small pieces to obtain the gene of interest. • These enzymes are also employed to weed out unwanted sequences from natural vector DNAs to create active vectors. • These enzymes are employed to split a large DNA into small pieces for nucleotide sequencing. • They are employed to generate a restriction map of DNAs. • They are employed to identify variations of close individuals using restriction fragment length polymorphism (RFLP). 11.5 DNA LIGASES – JOINING DNA MOLECULES The DNA ligase is basically a “molecular adhesive.” It provides the tools for cutting and assembling DNA molecules with REs. DNA Ligase is an

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enzyme joining the ends of two duplex DNAs to generate a long DNA. It cannot add nucleotides to fill the gaps in the DNA. It fills the gap by drawing up a covalent bond between the 5’-phosphate group and the 3’-OH group in the gap. This covalent bond formed between the 5’-phosphate group and the 3’-OH group, which fills the gap, is known as a phosphodiester bond. If one or more nucleotides are missing or 5’-phosphate group is absent in the gap, and then the gap is never sealed by the enzyme. This function is critical for the accomplishment of many experiments, making the DNA ligase a key enzyme in genetic engineering. DNA ligases were first discovered by Gellert, Lehman, Richardson, and Hurwitz in 1967 (Shuman, 2009). The most popular DNA Ligase derived from T4 bacteriophage – a monomeric polypeptide with a MW of 68,000 Daltons is encoded by bacteriophage gene 30. This enzyme is not effective only at sealing gaps in fragments that are held together by cohesive ends, it is also effective in joining blunt ended DNA molecules under appropriate conditions. In order to prevent thermal denaturation of the short base-paired regions that hold the cohesive ends of DNA molecules together, this enzyme is often used at much lower temperatures (4–15°C) (Shuman, 2009). 11.5.1 USES • DNA ligase is used to link a vector DNA to a target DNA to construct rDNA; • It is used to assemble the DNA fragments of two different organisms to create vectors with desired characters; • It is can add linker and adaptor sequences to blunt ended vector DNA and target DNA; • It is used to combine oligonucleotides into the chemical synthesis of DNA by ligase chain reaction. 11.5.2 REVERSE TRANSCRIPTASE Reverse transcriptase is an enzyme that uses mRNA as a template to create a DNA strand. It is also referred to as RNA dependent DNA synthetase. It functions by adding complementary deoxyribonucleotides onto the mRNA template to generate a complementary DNA strand. This enzyme (Molecular Weight = 70,000 Daltons) is bound with the RNA core of the virus. The 3’-OH group required by the enzyme to add the first complimentary base to mRNA is provided by a primer.

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This enzyme is isolated from retroviruses. The examples of retroviruses from which the enzyme is isolated include Rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), murine leukemia virus (MuLV) and avian myeloblastosis virus (AMV). 11.5.3 RIBONUCLEASE-H

Ribonuclease-H is a type of nuclease enzyme that hydrolyzes mRNA in RNA-DNA hybrid and that mRNA is used to synthesize cDNA. It is isolated from retroviruses. These include RSV, MMTV, AMV, etc. RNase – H (MW = 92,000 Daltons) is found in association with reverse transcriptase enzyme in the RNA core of the viruses. 11.5.4 ALKALINE PHOSPHATASE The enzyme alkaline phosphatase digests the terminal phosphate group from 5’-end of a DNA fragment. The enzyme is made of two identical subunits. It has a molecular weight of 1,40,000 Daltons. This enzyme also has 4 zinc atoms. It prevents self-annealing of vector DNA by removing the 5’-phosphate group from its linearized state, while making rDNA. 11.5.5 POLYNUCLEOTIDE KINASE Polynucleotide kinase (MW = 34,000 Daltons) transfers a phosphate from ATP to 5’-OH group of dephosphorylated DNA or RNA. This enzyme is made up of four identical subunits. 11.5.6 DNA POLYMERASE DNA polymerase is a complex enzyme. It synthesizes nucleotides complementary to the template strand. It adds nucleotide to release the 3’-OH end and helps to lengthen the strand. It also fills the gaps in double stranded DNA. DNA polymerase-I which is isolated from E. coli is frequently used in gene cloning. Taq DNA polymerase (MW= 95,000 Daltons) isolated from gram-negative, rod-shaped bacterium, Thermus aquaticus, is used in polymerase chain reaction (PCR).

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11.6 CONSTRUCTION OF RECOMBINANT DNA (RDNA) rDNA as discussed earlier is the hybrid DNA that has been tailored artificially by combining DNA fragments of two different organisms. rDNA is briefly called rDNA. It is an altered sequence of gene. In RDT, a desired gene is transferred to a vector, i.e., plasmid. The vector containing the desired gene is called the rDNA. It is introduced into a host cell, for example, E. coli. The E. coli containing rDNA is called a recombinant organism. When E. coli multiply, our gene of interest also replicates, producing multiple copies of the desired gene. This is called gene cloning. Steps involved in the construction of rDNA: 1. Gene Library: Before cloning any gene of interest with a viral or plasmid vector, it is very important to build a DNA library. A DNA library is a collection of cloned DNA fragments, including our target gene. A DNA library is usually maintained in a large number of bacterial cells. Genomic and cDNA are two important forms of DNA libraries. The cDNA library represents a collection of only those DNA fragments that were transcribed into mRNA in the cell from which the mRNA was isolated. 2. Genomic Library: It refers to the entire genomic setup of the individual from which the DNA was created. A genomic library is framed from chromosomal DNA. First, all DNA is digested by specific enzymes called restriction nucleases to produce large pieces of DNA. The reaction is done under controlled conditions to produce DNA fragments of about 20 Kbps. The advantage of a partially digested library is that it contains a series of overlapping DNA segments spanning the genome. This is followed by the fragmentation of DNA according to size, as the size of the genomic DNA fragments is very large. The second step involves cleavage of bacteriophage λ DNA with exactly similar restriction nuclease that was previously used to digest genomic DNA. The two DNA are then mixed by the addition of DNA ligase. The chimeric DNA is enclosed in the phage capsid. The final phage capsid is permitted to infect bacteria, thereby allowing multiplication of recombinant phage as the bacteria grow in number. This whole process generates millions of genomic DNA clones.

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After purification of phage, virions are collected to form a phage genomic library. Genomic libraries are typically kept as phage particles in solution.

A genomic library can also be organized in cosmid vectors by adjusting the DNA fragment size to 40 to 45 kb after partial digestion. A versatility of the cosmid library lies in its larger insert size, but there are high chances of rearrangement also. In recent times, scientists have constructed YAC libraries to map large inserts of the human genome (Jen & Travers, 2013). 3. cDNA Library: It is constructed from specified mRNA molecules. If the expression of the gene of interest is elevated, the majority of cDNA clones are possibly containing the gene sequence, thus cDNAs can be drawn from these cells very easily. However, if the gene of interest is not transcribed in abundance, enrichment of specified mRNA is done by various methods to construct the cDNA library from that cell. The best example is the application of antibody against protein for precipitating only selected polyribosomes to which coding mRNA is attached. The precipitation enhances the required mRNA by almost 1000-times. Isolation of mRNA is done by dissolving cells in solution, which arrests the activity of ribonucleases followed by the separation by cesium chloride density-gradient centrifugation. mRNA (contains poly-A tail) gets detached from rRNA and tRNA by subjecting through cellulosic chromatographic column covalently attached with the short polymer of thymidine. Due to the presence of adenines at the 3’-terminal of mRNA, mRNA binds to oligo-dT and is therefore taken by the column resulting in only poly-A RNA (rRNA and tRNA) passing through. Elution of pure poly-A plus mRNA is done by washing it with water followed by the mRNA conversion into ds DNA by subjecting to the series of enzymatic reactions. Firstly, the hybrid molecule contains a single strand of RNA, and the other strand of DNA is prepared with the help of reverse transcriptase. By application of DNA polymerase, DNA ligase, and RNase H, the RNA strand is then changed into DNA resulting in the formation of cDNA molecule. The second molecule of cDNA is added to the first one with the help of Klenow DNA polymerase. This double-stranded DNA is synthesized from one molecule of cDNA which is of the same length as that of initially used mRNA. The final cDNA is

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identical to a gene that codes from mRNA in prokaryotes but contains non-coding introns in the case of eukaryotes which ultimately interrupts the coding sequence. The resultant cDNA molecule is joined into a cloning vector (e.g., phagemid, eukaryotic vector, λ phage) to produce the cDNA library (Singh et al., 2021). In order to proceed with gene cloning, the insert DNA is isolated from the recombinant organism and spliced with REs. The same is then separated by electrophoresis, amplified, and ultimately use for amplification of the gene. 11.7 INTRODUCTION OF FOREIGN DNA FRAGMENT INTO A VECTOR The DNA thus isolated as in above is disintegrated with the help of REs to produce the cohesive ends. It’s important to treat cloning vectors (e.g., pBR322) with similar REs to generate complementary cohesive ends. The plasmid contains ampicillin and tetracycline resistance genes against antibiotics. However, in this process, some important genes are also destroyed by the REs in the last product. For instance, Bam HI destroys terr gene. The sticky ends can thus permanently join if the addition of T4 ligase with ATPs at 4–10°C for prolonged incubation time is done. This reaction produces a heterogeneous mixture of various products including recombinant molecules and parental plasmids. For insertion of ds cDNA in any vector, it is essential to produce sticky ends in an ssDNA sequence opposite to DNA present at the end of the linearized vector. The two important methods to generate cohesive ends in double-stranded DNA include the use of restriction enzyme linkers and the use of homopolymer tails. 11.8 TRANSFER OF RECOMBINANT DNA (RDNA) INTO BACTERIAL CELL Various methods have been devised for introducing rDNA into host cells primarily depending upon the nature of vector and host. Some of the methods are discussed below: 1. Transformation: First described in 1928 by Frederick Griffith, transformation is a process by which free DNA is internalized by another cell. The transformation includes binding donor DNA to the capable recipient cell followed by the entrance of this

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DNA. This DNA penetrates the other cell through an unspecified mechanism. Ultimately, this DNA gets incorporated into the chromosomal DNA by homologous recombination (Hanahan, 1983; Bergmans, Van, & Hoekstra, 1981) (Figure 11.4).

FIGURE 11.4

Principal steps in transformation.

2. Chemical Transformation: In this method of chemical transformation, the cells are allowed to grow till the middle of the log phase, harvested, and then subjected to divalent cations such as CaCl2 treatment to make them capable. Afterwards these capable cells are mixed with DNA at low temperature followed by minute heat shock at 42°C. Before plating, cells are incubated with a rich medium for 30–60 mins on LB agar plates containing antibiotics. There is no need for specialized equipment for transformation as well as no need to remove salts from the DNA in this method of transformation (Figure 11.5).

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FIGURE 11.5

Chemical transformation by CaCl2.

3. Electroporation: It is also known as electropermeabilization. It is a technique to increase the permeability of cells by the application of an electric field. In this method of transformation, the cell is allowed to grow till the mid-log phase, followed by extensive washing with water to remove all salts from the media. The glycerol is added to the water up to 10% of the total concentration to keep the cell stored at freezing temperature for future use. Electroporation is done on a mixture of E. coli and interested DNA in a plastic cuvette supplied with electrodes. A tiny electric pulse of 2400 volts/cm is supplied to cells which creates small pores in the membrane. The target DNA enters through these pores. Afterwards, the mixture is incubated with broth and plated. For electroporation, it is important to have salt free DNA (Ozyigit, 2020).

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11.9 DNA DELIVERY IN MAMMALIAN CELLS The cellular structure and makeup of mammalian cells are different from the prokaryotic cell. Keeping in view the surface chemistry and internal structure of mammalian cells, the uptake procedure also will be different in these cells resulting in the development of new specialized methods. There are six major strategies of DNA delivery in mammalian cells: 1. Chemical Transfection Techniques: The technique is based on the principle of coating DNA or forming complex with polymeric compound up to the extent that it may easily precipitate. The complex formation or coating makes possible the interaction of the precipitate with the membrane, thereby internalized through endocytosis. Various chemical compounds have been discovered, which possess the property to make complex and deliver DNA inside of the cell (Figure 11.6).

FIGURE 11.6

Chemically mediated DNA delivery in animal cell.

2. Calcium Phosphate Method: This technique though easy is very inconsistent due to its overall dependence on DNA-phosphate complex size and its difficulty to control. This method includes the mixing of calcium chloride and DNA in phosphate buffer followed by incubation of around 20 minutes. The resultant mixture is supplemented on a plate in a drop wise manner. This

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final DNA-calcium phosphate complex ultimately gets precipitated and layered on the cell uniformly and is internalized by the cell via endocytosis. Inside the cell, DNA escapes from the precipitate and travels to the nucleus by an unknown mechanism. This method is suitable for those cells which grow in monolayer or suspension and not in the case of cells that grow in clumps. 3. Polyplexes Method: Due to the deposition of particulate matter on the surface of the cell, it loses its cellular integrity due to physical abrasion. This causes increased cellular toxicity and reduced cell viability. To avoid this, a new method, namely the polyplexes method was devised. In this method, DNA forms a complex with a chemical agent in the form of soluble precipitate (polyplexes) via electrostatic interaction. Various chemical agents used include positively charged cationic lipids (transfection), polycationic carbohydrates (DEAE-Dextran), and polyamines (polyethyleneimine). The final soluble aggregates of DNA with the polycationic complex are quickly internalized by the cell thereby to the nucleus for expression (Figure 11.7).

FIGURE 11.7

Mechanism of polyplexes.

4. Liposome and Lipoplex Method: In this method, DNA is packed in a lipid vesicle or simply liposome. The vesicle containing the DNA is allowed to fuse with the cell membrane and thereby deliver

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the DNA into the target cell. Transfection efficiency depends directly on the preparation and encapsulation of DNA. Cationic and neutral lipid formed liposomes when bound with DNA are called Lipolex. This method is usable in a wide range of cells and is proven to transfect larger DNA size and can be used to target specific sites in the cell (Balazs & Godbey, 2011). 5. Bactofection: This method is very accepted in plants where Agrobacterium tumefaciens is used. In this method, bacteria upon entry get lysed inside the phagosome and release their DNA in the cytosol. Commonly used bacterial species in bactofection are salmonella, shigella, etc., in most the case, these strains are firstly attenuated to render them harmless (Palffy et al., 2006). 6. Transduction (Virus-Mediated): Viral particle has a unique and indigenous tendency to infect and carry DNA inside eukaryotic cells. Cloning gene of interest into the viral vectors is a novel way of delivering this DNA into the host cell. The availability of recombination sequences results in the integration and replication in the host respectively. Since, viruses possess vital machinery for protein expression required for DNA replication, RNA polymerase, and other ligands for attachment on the host cell. Additionally, it possesses other structural components to regulate the infection cycle (Giacca & Zacchigna, 2012). 11.10 SCREENING OF RECOMBINANT CLONE As seen above multiple vectors are used in cloning to construct a clone. After the transformation of the clone into a proper host, it will produce colonies. These colonies are subjected to need screening to identify clones containing the desired gene fragment. 11.10.1 CHROMOGENIC SUBSTRATE This is one of the easy and simple methods of screening and is based on the principle of color change. In this method, a chromogenic substrate is used to detect a particular enzymatic activity that will be present or absent in the clone. The most popularly used system that uses this feature

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includes “Blue-white screening.” X-gal or 5-bromo-4-chloro-3-indolyl-βD-galactoside is the colorless substrate used in this method. X-gal is a substrate of the enzyme β-galactosidase which in turn is the product of the lacZ gene of the lac operon. β-galactosidase is a tetrameric protein and activity lies in the N-terminal region (11–41). If the foreign gene is inserted into the lacZ region, the gene will be inactivated and hence no β-galactosidase is synthesized. Thus, host cells containing rDNA will form white colored colonies on the X-gal plate. In the case of nonrecombinant cells, the lacZ gene will be intact producing β-galactosidase. β-galactosidase acts on X-gal to form an insoluble, blue-colored product (Maas, 1999). Hence, blue-colored colonies designate the absence of insert whereas colorless colonies indicate the presence of an insert (Figure 11.8).

FIGURE 11.8

Blue–white screening.

11.10.2 INSERTIONAL INACTIVATION METHOD It is a very efficient method and involves the disruption of one of the genetic traits by inserting foreign DNA. Based upon the genetic trait disrupted there are various approaches in this method. 11.10.3 INSERTIONAL INACTIVATION OF ANTIBIOTIC RESISTANCE GENE As described earlier, bacterial plasmid pBR322 has ampicillin and tetracycline antibiotic resistance genes. If we clone the gene in the ScaI region of Pbr322, it will disrupt the ampicillin resistance, but the tetracycline gene will be intact. To select and screen the clone, the transformed E. coli is first

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plated on a tetracycline-containing medium (Bal, Cegłowski, & Maciag, 1982). Similarly, a replica plate is made in which media is supplied with tetracycline but not ampicillin. 11.10.4 ANTIBIOTIC SENSITIVITY

This is a simple method of screening recombinants. Traditionally, a circular plasmid contains antibiotic resistance genes, and growing the clone on antibiotic supplemented media will not cause any effect on the clone. However, when the circular plasmid is digested with REs and fragment is ligated to give back circular plasmid with insert. Now both circular clones and cut plasmid into host are allowed to grow media supplemented with an antibiotic. Only circularized clones with intact antibiotic resistance regions will give colonies whereas cut plasmid will not grow (Figure 11.9).

FIGURE 11.9 Antibiotic sensitivity.

11.10.5 COMPLEMENTATION OF MUTATION The yeast strain is very handy in screening recombinant clones as it has four different genes, i.e., His3, Leu2, Trp1, and Ura3 which can be used as a selectable marker. These genes can be mutated to screen the recombinant clone. However, Ura3 and Lys2 markers offer both positive and negative selection. Positive selection: In this method, vectors express a lethal gene, like a restriction enzyme that digests the bacterial genomic, when DNA is successfully inserted in the plasmid making the lethal gene useless. Therefore, only cells containing recombinant plasmids are able to grow. Negative selection-In this, a compound is supplemented to the media, and

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it is transformed to the cytotoxic compound in the presence of a gene product. This ceases the growth of wild-type cells. However, recombinant host strain has a non-functional gene product and grows in the same media. Additionally, there are various other methods and strategies that are in the current trend to screen the recombinants from wild type. 11.11 PROTEIN PRODUCTION STRATEGIES IN EXPRESSION SYSTEM After successful recombination, it is important to choose an ideal protein expression system to get maximum yield. Heterologous expression systems can be very useful as they provide a very cheap and increased output of the proteins for applications. Currently, a variety of eukaryotic and prokaryotic expression systems are used for this purpose, each having its unique feature. The most commonly used E. coli system is very costeffective and can be easily manipulated but has its limitations as well. This system cannot express proteins with post-translation modification. However, the eukaryotic expression system is very efficient to perform post-translational modifications but demands a very high cost. 11.11.1 CRITERIA TO CHOOSE AN EXPRESSION SYSTEM Selection of a host expression system is the first phase in deciding on strategies for cloning the gene in a suitable vector and following downstream procedures. The number of factors needs consideration while choosing the appropriate host-expression vector system for overexpression of a protein: 1. Quantity of the Desired Protein: If a small amount of protein is required, any host expression system may be suitable, but if large amounts of protein are required, an E. coli or yeast expression system may be more appropriate than the mammalian expression system. 2. Size of the Protein: For large size of the protein eukaryotic expression system is preferred over E. coli expression system. 3. Compatibility between Source Organism and Expression System: Generally, a close distance between the source organism

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and the expression system is preferred, as it can raise the chance of obtaining expression of the cloned gene and the existence of the protein in the soluble fraction. 4. Down-Stream Application: This is the most important yardstick for choosing a host-vector system. If protein production is for making antibodies, any expression system may be suitable for this purpose, but if the protein is required for activity or ELISA, a compatible expression system is preferred. KEYWORDS • • • • • •

clustered regularly interspaced short palindromic repeats genetic engineering principles genetically modified site-specific nucleases transcription activator-like effector nucleases zinc finger nucleases

REFERENCES Bal, J., Cegłowski, P., & Maciag, I., (1982). Construction of plasmid vectors for gene cloning in Escherichia coli and Bacillus subtilis. Acta Microbiologica Polonica, 31(3, 4), 217–225. Balazs, D. A., & Godbey, W., (2011). Liposomes for use in gene delivery. Journal of Drug Delivery, 2011. Bergmans, H., Van, D. I., & Hoekstra, W., (1981). Transformation in Escherichia coli: Stages in the process. Journal of Bacteriology, 146(2), 564–570. Britannica, T.E.O.E., May 18. Restriction enzyme. Encyclopedia Britannica. https://www. britannica.com/science/restriction-enzyme) (accessed on 14 October 2022). Giacca, M., & Zacchigna, S., (2012). Virus-mediated gene delivery for human gene therapy. Journal of Controlled Release, 161(2), 377–388. Hammer, Gary D., Stephen J. McPhee, & McGraw-Hill Education, eds. (2014). Pathophysiology of disease: an introduction to clinical medicine. McGraw-Hill Education Medical, 2014. Hanahan, D., (1983). Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology, 166(4), 557–580.

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Hohn, B., & Murray, K., (1977). Packaging recombinant DNA molecules into bacteriophage particles in vitro. Proceedings of the National Academy of Sciences, 74(8), 3259–3263. Jen, K. Y., & Travers, A., (2013). DNA-binding proteins. In: Brenner’s Encyclopedia of Genetics (2nd edn., pp. 345–347). Elsevier Inc. Maas, S., (1999). Efficient and rapid procedure for blue-white screening of recombinant bacterial clones. BioTechniques, 27(6), 1126–1128. Nicholl, D. S., (2008). Genetic Engineering (3rd edn.). Ozyigit, I. I., (2020). Gene transfer to plants by electroporation: Methods and applications. Molecular Biology Reports, 47(4), 3195–3210. Palffy, R., et al., (2006). Bacteria in gene therapy: Bactofection versus alternative gene therapy. Gene Therapy, 13(2), 101–105. Roberts, R. J., & Murray, K., (1976). Restriction endonuclease. CRC Critical Reviews in Biochemistry, 4(2), 123–164. Shuman, S., (2009). DNA ligases: Progress and prospects. Journal of Biological Chemistry, 284(26), 17365–17369. Singh, P. K., et al., (2021). From gene to genomics: Tools for improvement of animals. In: Advances in Animal Genomics (pp. 13–32). Elsevier.

CHAPTER 12

Genetically Modified Organisms: Concerns and Biosafety

MUHAMMAD ISHTIAQ, MUBASHIR MAZHAR, MEHWISH MAQBOOL, and MUHAMMAD WAQAS MAZHAR Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur, Azad Jammu and Kashmir (AJK), Pakistan

ABSTRACT Genetically modified organisms (GMOs) are those organisms in which some foreign genes are introduced from some other organisms to introduce beneficial or desired characteristics. The organism from which the genes are taken are called donor organisms, whereas the organism in which these genes are introduced are called recipient or transgenic organism. The organism or substance that introduces the foreign gene from the donor to recipient is called vector usually it is an organism like bacteria or some small viruses. Although GMOs are better producers of yield as compared to the wild type yet there are biosafety threats about the use of GMOs, so these organisms need to be carefully reviewed before the launching into environmental management systems. There are several challenges and concerns associated with the application and uses of GMOs, like environmental, ecological, and social concerns. There are several economic concerns as well as ethical concerns. More importantly, health and regulatory concerns are associated with the application of GMOs.

Genetic Engineering, Volume 2: Applications, Bioethics, and Biosafety. Tariq Ahmad Bhat & Jameel M. Al-Khayri (Eds.) © 2023 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)

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12.1 INTRODUCTION Genetically modified organisms (GMOs) have desired genes which are required for the production of desired characteristics in individuals. As these organisms have desired characteristics, and at the same time, there are some concerns related to their application. The concerns related to GMOs are economic concerns that it will create more monopolies environment, there are some other economic social and environmental as well as ecological issues are concerns related to their application (James, 2013). Most important of all concerned is human health at the same time there are some organisms that are being affected adversely with the application of GMOs (Ankita et al., 2016). As the population of the world is increasing day-by-day it is estimated that in 2050 the population of the world would be doubled as it is today but to feed such a huge population scientists need to introduce GMOs which can produce more yield as compared to the natural wild type GMOs as discussed earlier are beneficial for the production of better yield and less capita is used for their production (Moghissi et al., 2018). Biotechnology has enabled the scientist to create such species which have desired genes which will produce desired characteristics these genes are first of all identified in their wild type organisms then by biotechnological protocols these are isolated and carefully transferred into particular organism in which we want to see such characteristics (Kumar, 2012). Today modern world countries are greatly using and depending upon GMOs for the production of crops like wheat and other dairy products. Some of the modified fruits and crops have better yield, and they have less dependence on the herbicides and other pesticides for the removal of pathogens and pests, so they are better modified to cope with abiotic stress as well as biotic stresses. They can also produce growth promoters, insulin, and other special proteins (Prabhu, 2009). There is no doubt that GMOs have better characteristics in the sense of color taste texture and other characteristics but at the same time they also process a concern with ecology, sociology, moral and ethical values of the particular area in which they are sown (Devos et al., 2009). 12.2 GENETICALLY MODIFIED ORGANISMS (GMOS) AND CONCERNS RELATED TO THEM The GMOs have been faced by different challenges which are linked with their use and health of mankind. These GNOs not effect for human health

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but also these are linked with injurious impacts on habitat, ecology, social issues and environment as general. The GMOs have also been creating economic and ethical issues for the indigenous and various ethical or religious communities. The various health agencies are showing serious concerns on use of GMOs (EFSA, 2011). 12.2.1 CONCERNS RELATED TO ENVIRONMENT Environmental challenges related to GMOs can be classified into two classes. In one-way GMOs can give such things which are not good for the human and for the use of other organisms but on the other hand GMOs can also cause the elimination of certain genes and also, they can enhance the special characteristics into some other species which are related to them (Clark et al., 2006). Introduction of special genes into transgenic organisms may lead to changes in the gene pool of their relative species and it can also be transferred to the natives or the wild type organisms nearby. It can have severe consequences so the use of GMO must be very much regulated and there must be a specific protocol which must be followed and if these protocols are not followed the use of GMOs are not admissible (Robert & Guillermo, 2014). 12.2.2 CONCERNS RELATED TO ECOLOGY Ecological challenges related to GMOs include human beings and other negative effects on the other organisms, which are known as non-target organisms. These non-target organisms can also be affected by the gene introduced into transgenic organism so these can produce such substances which can have adverse effects on human health as well as the organisms which can consume these foods. In most of the cases these organisms which are known as transgenic organisms benefit the human race but, in some ways, they can produce negative effects not only on the genes of the organism but also on the health of the organisms which may consume (Leite et al., 2000). Due to the application of pest-resistant in GMOs large number of insects and bees are being affected adversely, and their population have decreased to an extent of alarming situation. One such example is the pathogenic bluetongue virus (BTV) also known as BTV it was introduced in European States due to the cultivation of a large number of transgenic crops (Fischer et al., 2004).

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12.2.3 CONCERNS RELATED TO SOCIETY As the field of biotechnology is advancing day by day but more of the people in developed countries as well as in developing countries have concerns about the application of these transgenic organisms, they think that it means the nature has been disturbed in some ways so these transgenic organisms may have consequences and nature may have some adverse effects on their health and on their wealth. Some of the analysts think that it will take 10 to 15 years to develop a scientific approach in developing countries and a large number of populations in developed countries also strikes behind (Giddings et al., 2000). Australia USA UK and European States have been utilizing several GMOs but the people living there are concerns related to their application. Most of the products that were produced by the GMOs in these states were not consumed in Europe just due to the concerns that people think nature must not be disturbed at any cost. A large number of populations demand a specific tag or label on the GMOs’ products so they can choose among the products which are not modified (Horn et al., 2004). 12.2.4 CONCERNS RELATED TO ECONOMY GMOs face economic challenges as the products from the GMOs and their prices would be much higher. It is thought that some of the companies which will own the license of production of GMOs will create market monopolies and they will cost a large amount in the market (Ankita et al., 2016). Economic concerns related to the application of GMOs and their availability in the market will be worsened by the corrupt officials of developing countries because they will try to store the products which are desired by the large number of population and will create false shortage in the market and hence, they will demand much higher prices as compared to their original price (Gruère et al., 2007). 12.2.5 CONCERNS RELATED TO ETHICS There are ethical concerns related to the release of GMO. Their approval and application must be carefully watched by the authorities. It is thought by a number of people that it will have adverse consequences on the other organisms which are present in nearby environment and ecological systems. They think that introduction of a few genes from other organisms

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into a particular organism means disturbing the nature’s production and it will have adverse consequences (Moghissi et al., 2018). 12.2.6 CONCERNS RELATED TO HEALTH As we know that there are social-ecological ethical and environmental concerns related to the use of modified organisms, but most important concerns related to the application of these transgenic organisms are health concerns (Phillips et al., 2000). It is noted by several researchers that GMOs and the use of their products have adverse effect on human health. GMOs have antibiotic resistance it may also cause antibiotic resistance in human by using such organisms or their products (Beckmann et al., 2006). In South Korea, China and Japan skin allergy was found to be associated with the use of genetically modified crops including maize and wheat. Several workers which were working in the field of cotton were affected adversely and it also have drug-resistance from a large number of available antibacterial drugs which have no effect on these patients (Sinemus, 2008). 12.2.7 CONCERNS RELATED TO REGULATION There are regulatory concerns related to the use of GMOs. As all the nations need to enhance their production by use of less capital. But their use must be carefully regulated by the law and regulatory authorities because if these organisms are allowed to leave the specific area it may disturb the production of other crops which are sown in this area. Most of the developing countries are being ruled by corrupt politicians and these politicians are on the verge of making money (Dibden et al., 2013). They are not interested in the welfare of their populations, and they are also ignorant of GMOs as well as the consequences that are associated with the application and approval of these products. So, it is estimated by the scientist that the use of GMOs in third world countries may have very adverse effects on the global population (Jones et al., 2008). 12.3 BIOSAFETY MEASURES GMO have some concerns related to their use in environment and it can also have adverse effects on health of human being and other organisms

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which may consume these products originated from the GMOs. When it comes to eliminate or reduce the possible negative effects of GMOs then it is now known as biosafety regulation. Biosafety means the procedures, principles and governmental policies which are ensured to be adopted for the conservation of environmental ecological and health safety of the organisms using these products as well as for human beings (CSBD, 2004). The main procedures and principles which must be shown for biosafety of other organisms from GMOs are discussed in subsections. 12.3.1 TO AVOID COMPETITION WITH OTHER SPECIES GMOs have better growth and development so it is thought that these organisms can have faster growth as compared to the wild types or the native organisms in that particular area. These GMOs may be invasive to a certain area, and it will have bad effects on habitats or native organisms both including plants and animals. GMOs should be very carefully monitored for their spread and growth so that to avoid the competition with natural species (Halford & Shewry, 2000). 12.3.2 DECREASE IN SELECTION PRESSURE ON NON-TARGET ORGANISMS As GMOs have better properties are characteristics that is why they can have better selection and the other organisms present their may lose their selection so it must be carefully regulated that GMOs may not compete for selection with other non-target organisms because it is very difficult for other organisms to compete with modified forms of characteristics that are only present in GMOs it will lead to distinct restricted populations of non-target organisms (Caiping et al., 2004). 12.3.3 MONITORING ECOSYSTEM IMPACTS The only few genes introduced into some modified organisms may have consequences not only limited to this CPC but it can also have some pressure and changes in other species which will be on the ecosystem sustainability so it is the need of time that GMO should be restricted to a specific

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ground or fields only. It must also be ensured to counter the impacts of this modification on that particular environment or ecosystem (Lee et al., 2003). 12.3.4 REGULAR FOLLOW UP The introduction of GMO will definitely have some issues related to the ecosystem and environment. So, scientist focus on regular follow-ups because it will lead to possibly pointing out the issues that are arising due to introduction of GMOs and the initial pointing out of problem is easy to be managed or eliminated from the ground. Regular follow-up is also needed to enhance the characteristics that are desired to be produced by modified organisms (Obert et al., 2004). 12.3.5 TO CHECK THE HORIZONTAL TRANSFER OF GENES

Whenever a GMO is introduced into an environment, it will possibly have a negative effect on the nearby populations of plants by transferring the genes into a horizontal manner that must be checked regularly. If the transgenic genes are transferred to other organisms that are non-target organisms it will produce disastrous effects because its impacts were not studied by the scientist. These changes due to horizontal gene transfer will have some possibly negative effects on human health and other organisms which are going to consume the products from this modified organism (Meli et al., 2010). 12.3.6 TO CHECK THE EFFECTS OF GENETICALLY MODIFIED (GM) PRODUCTS ON HEALTH OF CONSUMERS GMOs and the products that are originated from these organisms must be carefully studied for health problems or issues related to their application. It is necessary to carefully monitor the products originating from GMOs for the possible effects on human health. Once GMOs are introduced into the environment, it is not easy to eliminate these products in the market data regulated from these modified organisms, so scientists need to

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carefully observe the effects of these products on human health (Brookes & Barfoot, 2006). 12.3.7 PROPER MANAGEMENT AND REGULATION GUIDE Due to unawareness of people after approval and application of GMOs, situations are not able to be controlled, so when GMOs are to be launched in an environment there must be a comprehensive guide for controlling and regulating their products. Such guards will not only help the scientist to regularly check and monitor but also it will enhance the capital of farmers (Pusztai, 2001). 12.3.8 TO CONTROL UNINTENDED EFFECTS GMOs you really have some on intended effects on the environment ecosystem and other non-target organisms so before the approval and application of these organisms’ scientist should make a list of all possible unintended effects, and these effects must be presented before the regulatory authorities because if these organisms are introduced in environment of before knowing all the problems related to their application it will have consequences on other species (Ho, 2003). 12.3.9 MONITORING OF LONG-TERM EFFECTS Any change in some organisms’ genes will have long-term effects scientist need to carefully manage and regulate these effects. Great and careful studies should be done on these possible effects so that to make a where the farmer about the output of these modified organisms and to reduce the possible negative effects on human health as well as on the other species (Nuti et al., 1994). 12.3.10 TO RESTRICT FROM INVASIVENESS Scientists believe that GMOs can be modified in such a way that they can compete more effectively as compared to their wall types, and it is

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concerned that these modified organisms that they will become invasive and grow uncontrolled. Biosafety measures should be taken into account before launching GMOs to avoid the wilderness or invasiveness caused by these modified organisms into other habitats that are not target (Smith et al., 2010). 12.3.11 FIELD TRIALS CONFINED TO A PARTICULAR AREA

GMOs have the ability to grow into a large amount hence disturbing other populations, so scientists are allowed only to make this process or protocols only in a confined area to avoid the problem of invasiveness. Gene flow becoming a weed and other in Environmental impacts are also associated with the application of GMOs; hence if confined field trial is not used, it will lead to the problems of invasiveness and other ecological disturbances would be associated with their application (Devos et al., 2008). 12.3.12 CAREFUL MITIGATION OF TRANSGENIC ORGANISMS Scientist have found a problem to mitigate the problems associated with GMOs these are such jeans noun as mitigates. Search mitigated genes are introduced willingly into these modified organisms to avoid any problems of invasiveness associated with their escape from the trial fields, so these mitigated genes are able to reduce the population of the modified organism to only first and second generation after the generations of these modified organisms are not able to escape from the field strategy (Morris, 2002). 12.3.13 TRANSFORMATION STRATEGY OF CHLOROPLAST Chloroplast transformation is a strategy to prevent the horizontal gene flow from these modified organisms in such strategies the chloroplast genes are transformed and Dash these genes are not associated with the reproduction reproductive system so these genes are not transferred to the next generation and effect is only restricted to a plant and single generation in this way scientist are able to mitigate or reduce the problems of invasiveness. Bhai transformation in chloroplast genes scientist is able to restrict the gene flow that is the big problem associated with the application of GMOs but 100% of the gene flow cannot be considered (Altieri & Rosset, 1999).

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12.3.14 TO MAKE TRANSGENES IN STERILE MALES Horizontal gene flow can be reduced from GMOs to non-target organisms if trans change are made in male that are sterile to reproduce. Search mails are not able to transfer their genes to other organisms’ hands horizontal gene flow to the same CPC or to other non-target organisms can be prevented in much comprehensive way (CBAC, 2002). 12.3.15 INTRODUCTION OF APOMIXIS To avoid the problems that are associated with GMOs scientist need to introduce apomixis in transgenes because if apomixis takes place, there is no reproduction and no seeds are produced, so the next generation would not be affected and there would be no horizontal transgenes. Fruits are produced by pollination then the seeds will be produced it is possible to have the transgenes that are flown from the transgenic organisms, and the next generation would also have the transgenic abilities or characteristics (Rikki, 2008). 12.3.16 INTRODUCTION OF SELF-POLLINATION IN TRANSGENES While introducing the genetically modified crops plants scientist need to introduce a special process of self-pollination in such transgenes to avoid the horizontal gene flow search protocols and procedures have been adopted for rice and it was seen that the process of gene flow was successfully controlled and limited to the particular field only (Knight, 2010). 12.3.17 INTRODUCTION OF INDUCIBLE PROMOTER GENES From the genetically modified crops that are sown in a confined field, if scientist is able to remove the transgenes before the flowering of that particular modified organisms, then the gene flow and pollen flow both can be avoided. Search inducible promoter genes can be regulated before the flowering season and the transgene can be limited and muted. Such genes are able to produce the seeds that are sterile and not able to give

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the next generation hence the gene flow can be avoided specifically and successfully (Darabani et al., 2008). KEYWORDS • • • • • •

biosafety biotechnical advances biotechnology genetically modified microorganisms promoter genes transgenic organisms

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Devos, Y., Demont, M., Dillen, K., Reheul, D., Kaiser, M., & Sanvido, O., (2009). Coexistence of genetically modified (GM) and non-GM crops in the European Union: A review. Agron. Sustain. Dev., 29(1), 11–30. Devos, Y., Maeseele, P., Reheul, D., Speybroeck, L., & Waele, D., (2008). Ethics in the societal debate on genetically modified organisms: A (re)quest for sense and sensibility. Journal of Agricultural and Environmental Ethics, 21(1), 29–61. Dibden, J., Gibbs, D., & Cocklin, C., (2013). Framing GM crops as a food security solution. J. Rural Stud., 29, 59–70. EFSA Panel on Genetically Modified Organisms (GMO), (2011). Scientific Opinion on Guidance for risk assessment of food and feed from genetically modified plants. EFSA Journal, 9(5), 2150. Fischer, R., Stoger, E., Schillberg, S., Christou, P., & Twyman, R. M., (2004). Plant-based production of biopharmaceuticals. Curr. Opin. Plant Biol., 7(2), 152–158. Giddings, G., Allison, G., Brooks, D., & Carter, A., (2000). Transgenic plants as factories for biopharmaceuticals. Nat. Biotechnol., 18(11), 1151–1155. GM Crops-the Health Effects, (2008). Soil Association. www.soilassociation.org (accessed on 14 October 2022). Gruère, G. P., & Rao, S. R., (2007). A review of international labeling policies of genetically modified food to evaluate India’s proposed rule. AgBioForum, 10(1), 51–64. Halford, N. G., & Shewry, P. R., (2000). Genetically modified crops: Methodology, benefits, regulation and public concerns. Br. Med. Bull., 56(1), 62–73. Herb Barbolet, Ellen Desjardins, Denise Dewar, Conor Dobson, Robert Friesen, et al., (2002). Improving the Regulation of Genetically Modified Foods and Other Novel Foods in Canada. Canadian Biotechnology Advisory Committee, Report to the Government of Canada Biotechnology Ministerial Coordinating Committee. Horn, M. E., Woodard, S. L., & Howard, J. A., (2004). Plant molecular farming: Systems and products. Plant Cell Rep., 22(10), 711–720. James, C., (2013). Global Status of Commercialized Biotech/GM Crops: 2013. Brief 46: ISAAA: Ithaca, New York. Jones, K. E., Patel, N. G., Levy, M. A., et al., (2008). Global trends in emerging infectious diseases. Nature, 451(7181), 990–993. Knight, B., (2007). Agricultural Biotechnology in Europe. Crop protection monthly. Kumar, S., (2012). Biosafety issues in laboratory research. Biosafety, 1, e116. Lee, Y. S., Wetzel, E. D., & Wagner, N. J., (2003). The ballistic impact characteristics of Kevlar® woven fabrics impregnated with a colloidal shear thickening fluid. J. Mater. Sci., 38(13), 2825–2833. Leite, A., Kemper, L., Da Silva, M. J., & Luchessi, A. D., (2000). Expression of correctly processed human growth hormone in seeds of transgenic tobacco plants. Mol. Breed., 6(1), 47–53. Mae-Wan, H., (2003). Stability of All Transgenic Lines in Doubt. ISIS Report. Institute of science in society. Meli, V. S., Ghosh, S., Prabha, T. N., Chakraborty, N., Chakraborty, S., & Datta, A., (2010). Enhancement of fruit shelf life by suppressing N-glycan processing enzymes. PNAS, 107(6), 2413–2418.

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Index

A

proteins, 58

reactions, 57, 139, 190, 212, 223

Abiotic

Alpha-amylase, 195

resistance, 101, 102

Amaranthus, 80

stress, 24, 42, 90, 115, 119, 208, 210, 246

Amarasca biguttula biguttula, 36

resistance, 115

Amino acid, 12, 13, 38, 144, 147, 148

tolerance, 115

Aminopeptidases, 39

stressors, 210

Ampicillin, 234, 240, 241

AC4 gene-RNAi construct, 210

Anacystis nidulans, 85

ACC (1-aminocyclopropane-1-carboxAnimal

ylate), 211

biodiversity, 17

Adenovirus, 130, 134

husbandry, 169

Advanced trophic levels, 189

manufacturing methods, 144 Adverse exterior stipulations, 151

pancreas, 12

Agricultural

Plant Health Inspection Services

commodities, 19

(APHIS), 11, 63

pests, 186

transgenesis, 85

production, 28

Anthocyanin, 94

products, 7, 8, 186, 190

Anthropogenic activities, 2

resources, 109

Antibacterial resistance, 114

sciences, 205

Antibiotic, 61, 81, 83, 168, 222, 234, 235

seed banks, 189

compounds, 142

workers, 189

resistance, 15, 81, 83, 98, 99, 190, 192,

Agrobacterium, 25, 28, 55, 77, 92, 94, 136,

212, 240, 241, 249

239

genes, 212, 240, 241

mediated

markers, 15

genetic transfer, 94 tobacco plant, 78

transfer protocol, 92 Antibodies production, 134

tumefaciens, 77, 239

Anticancerous properties, 119

Agrochemicals, 24, 144

Antifreeze, 193 Agronomic

Antigens, 94, 95, 101, 138

performance, 206 Antihemophilic variables, 84

practices, 190

Antimicrobial treatment efficiency, 192 Albino rats, 99

Anti-nutrients, 215

Alkaline phosphatase, 231

Anti-nutritional effect, 198 Allergen, 15, 190, 195–197

Antioxidant activity, 115

Allergenicity, 57, 184, 192, 193, 196–198

Antisense

Allergic

RNA, 26, 27

assessment, 196

techniques, 93

hypersensitivity, 193

Aphids, 36, 37

260

Index

pesticides, 27

protein efficacy, 40 proteins, 189

technology, 35

Bacteria, 132, 135, 141

expression system, 12

genetics, 127

host cells, 222

Bacteriophage, 61, 73, 74, 82, 130, 225,

230, 232

Bacteriostatic, 114

Bacterium thuringiensis, 115, 120

Bactofection, 239 Basmati rice, 174

Basta herbicide, 210

Bean plant recombination, 58

Bemisia tabaci, 36, 115

Bemoaning, 135

Beneficiary genes, 55 Beta carotene, 77, 78, 137

desaturase, 78

production, 77

galactosidase, 240

Bhai transformation, 253 Bialaphos, 26

Binary fission, 128, 129 B Bioaffinity bioreporter sensor, 150 Bacillus, 24, 41, 44, 77, 92, 128, 137, 146, Bioaugmentation, 149, 151

Biochemical

164, 192, 208, 209, 216, 225, 227, 228

makeup (cytosol), 115

amyloliquefaciens, 225, 227, 228

mechanisms, 142

licheniformis, 146

parameters, 56

sotto, 24

pathway, 77

thuringiensis (Bt), 20, 23–27, 30–42,

44, 56, 57, 59, 64, 77, 78, 92, 97–100, Biochemistry, 126, 143

Biodegradable, 101, 141, 148–152

115, 137, 187–190, 192, 194, 196,

associated recombinant genes, 150

197, 208, 209, 212, 213, 216

plastics, 139

Bikaneri narma (BNBt), 39

cotton, 20, 23, 26, 27, 30, 33–40, 42, Bio-factories, 222 Biogeochemical cycles, 101

44, 56, 64, 78, 98–100, 137

Biohazard, 2, 53–56, 58, 61, 62, 185 crop feed rates, 56 products, 3

crops, 26, 41, 56, 190

recombinant DNA technology (RDT), formulation, 24 54

ingestion test, 41

controversy-recombinant DNA

israelensis, 24, 25

(RDNA), 54

microbial sprayed crops, 41

Apolygus lucorum, 37

Aquatic

ecosystems, 85, 188

microbial community, 61

Arabidopsis, 92, 94, 114, 115, 119, 208

thaliana, 94, 115, 117

Archaea, 127, 128

enzymes, 128 genetics, 128

species, 128

Arena virus, 4

Aroma rice, 174

Arthritis, 134

Arthropod abundance, 36

Artificial insemination, 130

selection methods, 206

Asexual fission growth phase, 129 Asthma, 223

Atherosclerosis, 223

ATP binding cassette gene-PgABCA2, 39 Attacin A, 114

Augmented photosynthetic activity, 84

Autoclaves, 4

Auxin, 13

Avian myeloblastosis virus (AMV), 231

Index effect of GM (non-targeted organisms), 59

genetically modified organisms (GMOS), 55

Biolistics, 131, 133

Biological

hazardous agents, 3, 4, 21

organism, 3

integrity, 2, 3, 16

Bioprocess change, 150

Bioremediation, 101, 102, 139, 140, 143,

149–152, 166

technologies, 151

Biosafety, 1–7, 9–15, 21, 23, 24, 55, 212,

213, 245, 250, 255

clearing house (BCH), 7, 21

measures, 249

apomixis, 254

avoid competition, 250

control unintended effects, 252 effects of genetically modified (GM)

products, 251

field trials, 253 horizontal transfer of genes, 251 inducible promoter genes, 254

make transgenes, 254

mitigation (transgenic organisms),

253

monitoring ecosystem impacts, 250

monitoring long-term effects, 252 proper management, 252

regular follow up, 251 restrict from invasiveness, 252 selection pressure (non-target organisms), 250

self-pollination in transgenes, 254 transformation strategy (chloroplast),

253

regulation, 250

Biosensors, 143

Biostimulation, 149, 151

Biotech markets, 18

Biotechnical advances, 102, 255

Biotechnological

advancement, 12

approaches, 1, 17

261

entities, 164

invention, 1, 18, 166

product, 1, 3, 6, 7, 10, 17–19

Program Under Toxic Substances Control Act, 62

protection, 18

protocols, 90, 92, 246

techniques, 89, 107, 169

Biotechnology, 2, 3, 7, 10, 15, 18, 40, 90,

92, 96, 102, 108, 111, 119, 136, 145,

148, 151, 161, 164–166, 183, 184, 186,

195, 205, 213, 221, 222, 229, 246, 248,

255

guidelines, 164

patent approval, 165

practices, 15, 186

Biotic

connections, 187

stresses, 92, 119, 246

Bivalent antimicrobial protein, 114

Blood

cells production, 171

chemistry, 196

coagulating factors, 173 Blue

berry plant, 173

colored colonies, 240

tongue virus (BTV), 96, 247 white screening, 240

Bollworm complex, 33–36

Bovine

growth hormone, 11, 12

somatotrophin (BST), 12, 13 Brassica oleraceae var. capitata, 209

Brazilian nut protein, 57 Breeding

programs, 112

technologies, 23, 108

Broad-spectrum

Bt crops, 30

insecticides, 37

synthetic insecticides dependencies, 35

toxicity, 24

Bromoxynil, 78

Brush border membrane, 38

Busseola fusca, 212

262

Index

C

Cadherin, 39

repeat 5,(CR5), 38

transmembrane mutation, 39

Calcium

chloride, 237

solutions, 133

phosphate, 133

method, 237

Cancer development, 54

Canola, 23, 28, 77, 188, 192, 194, 206

Capsids, 129

Carbohydrate metabolism, 111

Carbon dioxide

emission, 84

fixation, 84 Carcinogenicity, 42, 184, 191, 198

Carotenoid, 77, 94, 113, 187

Cartagena protocol, 6, 7, 76

Cas9 endonuclease, 214, 215

Catabolic pathway, 142, 143

Catalase, 95

Cattle breeding, 184

Cauliflower, 99, 115, 191, 208 mosaic virus (CaMV), 99, 191, 192,

208, 210

Cecropin B gene, 114

Cell

extraction, 132

nuclear envelope, 136

physiology, 74

signaling pathways, 115

to-cell communication, 115

Cellulose, 95

chromatographic column, 233

Chakrabarty invention, 179

Cheilomenes sexmaculatus, 36

Chemical

intertwining, 74

resistance, 209

transfection techniques, 237 transformation, 235 China National Intellectual Property

Administration (CNIPA), 179

Chinese cabbage, 114, 115

choysum, 115

Chloroplast

genes, 253

transformation, 253 Chromatography, 82

Chromogenic substrate, 239

Chromosomal

DNA, 232, 235

jumping, 80 Chronic

kidney disease, 212

replacement therapies, 64

toxic effects, 41 Chrysanthemum plants, 92

Chrysopids, 36

Chymosin, 135, 143, 144

protein, 135

Circular chromosomes, 128

Cisgenesis, 207, 215, 216

event, 215

Citrus sinensis, 118

Classic progressive nephropathy, 195

Clinical

allergy, 196

investigation, 193

Clonal, 10, 80, 82, 130, 133, 142, 146,

166, 223, 232, 234, 239, 242

aging, 129

gene, 239

properties, 140

vehicles, 82

Clustered

randomly interspaced short palindromic

repeats (CRISPR), 64, 79, 116, 118,

120, 131, 132, 136, 137, 147, 152,

207, 214, 215

genome editing, 118, 214

sequences, 147

regularly interspaced short palindromic

repeats, 243

CO2 effective photon flux, 111 Coastal flooding, 208 Codex Alimentarius Commission, 8, 196

Coleoptera, 25, 26, 30, 31, 190

Collybia velutipes, 80

Color taste texture, 246

Commercial, 78, 213, 215

application, 178

Index cost, 213

cultivation, 24

industries, 62

microbial formulation, 27 production, 79

propagation, 1

protocols, 213

riboflavin, 144 Common rice grain, 77

Comorbidities, 134

Complementary

deoxyribonucleotides (cDNA), 208,

230–234

library, 233

sticky ends, 227

Computer codes, 175

Conjugation, 127 Conservative breeding procedures, 75

Constitutional institutes, 179

Contaminated ecosystems, 84

Convention

biological diversity (CBD), 6, 8, 21

breeding, 23, 25, 108, 206

food crops cultivars, 212 International Trade in Endangered

Species (CITES), 20 methods (agriculture), 91

Copy DNA (cDNA), 173

Copyright, 159, 175

protection, 2

violation, 175

Coronary artery angioplasty, 13

Cosmid, 82

library, 233

Cotton, 29, 30, 32–35, 99, 206

agroecosystem, 36

jassid, 36 population, 36

production, 32, 33

Cowpea, 26, 44

trypsin inhibitor (CpTI), 26, 36, 44 Creatinine plasma concentration, 195

Crop

adaptability, 110

breeding techniques, 109

clonal breeding (clonal selection), 109

hybridization selection, 110

263

introgression of genes (transgenic engineering), 111

mutation breeding, 110

photoautotrophic micropropagation,

111

plant breeds (molecular techniques),

112

polyploidy breeding, 110

speed breeding (accelerated plant

breeding tools), 112

tissue culture technique, 111

commercialization, 213 genomics, 112

improvement, 83, 109, 110, 205

infestations, 209 lineage, 109

production, 24, 27, 28, 30, 108

Cross breeding, 59, 109, 209

Cry

genes, 44

protein, 31, 40–42, 57, 64

Crystalline protein, 115

Cumbersome protein engineering, 214

Curcumin, 119

Custom catalysts, 147

Cytophaga sp., 146

D Damage-causing insect pests, 27

Danaus plexippus, 213

Decarboxylases, 95

Delta endotoxins, 187

Deoxyribonucleic acid (DNA), 10–12,

14, 25, 26, 28, 40, 53, 54, 57, 61–64,

73–76, 78–83, 107, 116–118, 120, 125,

127–134, 136, 139–141, 143, 145–148,

161, 165, 166, 170–172, 183, 184, 187,

192, 193, 197, 198, 207, 208, 214,

221–227, 229–241

calcium phosphate complex, 238

engineering technology, 120

ligases, 82, 131, 230

manipulations, 141

mutation, 132

phosphate complex, 237

polymerase 1, 82

Index

264 recombinant technology, 170

repair mechanism, 116, 117, 214

replication, 81, 239

sequences, 54, 79, 146, 165

Department of Biotechnology supervision, 30 Desirable mutations introgression, 110

Development

cloning vehicles, 74

program, 168

superweeds, 212

technology, 172

Diabetes, 11, 12, 138, 222, 223

Dietary consumption, 42

Differential screening, 80 Digestive

disorders, 191, 198

GI tract enzymes, 41 Diploid micronuclei, 129

Dipteran pests, 26

Disease

free crop, 134 resistance, 85, 112, 194, 210, 215

genes, 110

Disinfectants, 93 Dissemination mechanisms, 16

Distinctive uniform stable (DUS), 18 Documentation, 7

Dominant mutations, 117

Dormant

seeds, 98

soybeans, 98

Double haploid protocol, 112

Down-stream application, 243

Drought, 28, 29, 83, 85, 91, 92, 115, 186

resistant varieties, 28

Drug company, 171

E Earthworms, 59

Ecological disturbances, 253

Economic monopolies, 101

Ecosystem

impact quotient (EIQ), 35, 44

integrity, 212, 216

Ecotoxicological effects, 151

Eco-welfare organizations, 75 Edible vaccines, 77, 78, 134, 138

Effector nucleases, 120 Eggplant, 26, 97, 115

Electric shock treatment, 133

Electropermeabilization, 236 Electrophoresis, 207, 234

Electroporation, 74, 82, 86, 133, 134, 136,

236

technique, 133, 134, 136

Electrostatic interaction, 238

Embryonic stem cells, 131, 136

Endangered species, 20, 59

Endocytosis, 237, 238

Endogenous

DNA

biological repair system, 214

repair mechanism, 116

enzymes, 143 gene, 146, 148

protein concentrations, 196

toxins, 89

wild-type enzymes, 147 Endonucleases, 112, 117, 224–226, 228

Engineered

plants, 209

toxins, 188

Enolpyruvylshikimate-3-phosphate

synthase (EPSP), 210

Environmental

ethics, 216

fields, 140 hazards, 44 health, 27, 42, 95

integrity, 2, 16, 212

management system, 137, 245

microbiology, 151

perspectives, 76

pollution, 27, 74, 84

Protection Agency (EPA), 24, 40–42,

53, 56, 62, 197

resources, 83

risk assessment, 64

stresses, 91, 92

technology sector, 141

Erwinia uredovora, 77, 138

Erythropoietin, 171, 173

Index

265

Escherichia coli, 3, 12, 54, 76, 78, 95, 127,

131, 138, 150, 187, 222, 225–228, 231,

232, 236, 240, 242

bacteria, 12, 131

Essential

amino acid, 13

human hormones, 134

Ethidium bromide stain, 133

Ethylene precursors, 211

Eukaryotes, 81, 128, 130, 133, 234

European patent

convention, 198

office (EPO), 171, 179 Euschistus servus, 36

Exonuclease, 224

Experimental

medicines, 55

protocols, 223

Expression

coat protein genes, 26

endogenous genes, 21

External recombinant construct, 81

Extrachromosomal elements, 226

Extremophiles, 128

F Farm

community, 26, 34, 188

land biodiversity disturbance, 190

scale evaluations (FSE), 189, 216

Far-red light intensity, 113

Fecundity, 36, 37

Feed conversion efficiency, 195 Female mortality, 212

Fermentation, 143, 148, 164, 171

Field-evolved resistance, 39

Filamentous fungi, 128 Financial crisis, 40

Fish allergies, 193

Flavor saver tomato, 78, 107, 113, 206, 211

Floriculture, 93

Fok1 endonuclease, 117, 214

Follicle-stimulating hormone, 134

Follistim, 84

Food

Agriculture Organisation (FAO), 9, 41

allergy, 197

availability, 95

Drug Administration (FDA), 11, 12, 26,

140, 146, 148, 180

industry, 83

insecurity, 119, 120

processing, 57, 125, 134, 135

industrial environment, 57

security, 2, 9, 27, 89, 167, 205, 216

safety, 9 sustainable agriculture, 27

Foreign genes, 96

Forward genetics, 132

French High Council of Biotechnologies

Scientific Committee, 57 Freshwater dolphins, 59

Friedrich-Karl Beier patents, 164

Full-fledged human insulin, 222 Fungi, 128

G Galanthus nivalis (GNA), 194–196, 216

Gastrointestinal tract, 192, 193, 195

Gel electrophoresis, 80–82

Gene

amplification, 80 cloning, 28, 76, 80, 207, 223, 231, 232,

234

editing, 74, 79, 86, 147, 148, 214, 221, 222

proteins, 147

tools, 79, 214

evolution, 141

expression, 98, 100

frequencies, 16, 187 gun, 25, 28, 55, 131, 133, 136

library, 232

manipulation, 14, 64, 130, 132, 206, 215

packaging amplification, 28 pharming, 84

phenotype, 131

pyramiding, 37

regulation, 128

separation, 75

splicing, 54, 64, 170

targeting techniques, 136

therapy, 13, 14, 84–86, 126, 133, 134,

139, 140

applications, 140

costs, 86

266 transfer experiments, 78, 79 transference, 74 treatment, 75

General Electric Research Development

Center, 166

Genetic

altered, 96, 184

characteristics, 191

microorganisms, 84

organisms, 3

combinations, 53, 223

designed crop varieties, 76

diversity (GD), 108, 187, 216

elements, 132, 133

engineering (human welfare), 1, 4, 10,

23, 28, 30, 53–56, 64, 73–77, 79–81,

83, 84, 86, 89, 107, 108, 120, 125,

127, 130–132, 134–136, 140, 146,

147, 149–151, 159, 169, 170, 183,

185, 196, 205–211, 213–216, 221,

222, 224, 225, 230, 243, 245

animal improvement-transgenesis, 85

animal, 25

application (genetic engineering), 84

approval committee (GEAC), 30

benefits of, 83 biodegradation bioremediation, 148

challenging future prospective, 101 crop improvement-transgenesis, 85

crops, 42, 137, 140, 191

ecological challenges, 96

economic challenges, 97

electroporation, 79

environmental challenges, 95

ethical challenges, 98

food industry, 83 foods, 191 gems use (bioremediation), 150

gene delivering, 79

gene editing technique, 79

gene pool disturbance, 98

health challenges, 99

medicine, 84

metaphysical spiritual challenges, 100

microbes, 167

microorganisms (GEMs), 125–127,

141–144, 146, 148, 152, 184

Index organisms (GEOs), 25, 84, 135, 184

plants, 84, 138

principles, 243

process, 135

product, 11

recombinant DNA (rDNA), 79

regulatory challenges, 99

social challenges, 96

some drawbacks of gems (environment sector), 150

species, 185

technique (GET), 55, 89, 120, 131,

196, 205, 221

technology, 120

tomato plants, 113

type of techniques, 79 unintended impacts (other species),

100

guides, 147

improved crops, 76

information, 132, 222, 223 makeup, 2, 10, 25, 90, 98, 108, 130,

134, 144, 145, 152

maladies, 117

manipulation, 108, 139, 170, 194, 224

material, 55, 60, 61, 63, 74, 110, 127,

133, 136, 140–142, 170, 172, 221

modified (GM), (2–255) agriculture sector, 137

alteration in nutritional composition,

28

baculovirus, 61

Bt maize pollens, 213 cisgenesis-intragenesis, 214

climate change drought abiotic stress,

28

containing goods, 186

controversy (GMOS production), 139

crops, 27, 29, 44, 206, 249, 254

developed GM crops, 28

environmental management, 139

food security sustainable production,

27

genome editing technologies, 213

intervention, 2, 3

introduction, 135

medicine research sector, 138

Index microorganisms (GMM), 53, 54,

60–64, 148, 151, 166, 255

new trends, 213

organism (GMOs), 2–4, 6–10, 15,

21, 25, 30, 53–55, 60, 64, 73–76,

83, 86, 89–102, 131, 135–140,

146, 148, 152, 165, 167, 169, 180,

184–187, 190, 191, 194, 197, 198,

221, 245–254

pesticidal consumption-health

hazards, 27 production (GMO), 136

products, 30, 96, 97, 99–101, 140,

190, 191, 212, 216

Pseudomonas putida, 61

regulations, 95

technologies, 189

recombination, 54, 126, 129

research, 83, 128, 140

resources depletion, 108

screening, 132

systems, 143

variability, 109

Genome, 2, 10, 14, 54, 75, 78, 79, 82, 90,

98, 101, 112, 116–120, 127, 135, 136,

146, 207, 210, 214, 215, 232, 233

editing, (116–215)

clustered randomly interspaced short

palindromic repeats (CRISPR), 118

procedures, 117

projects, 120 technologies, 116, 214

tools, 116–120, 214, 215

transcription activator-like effector

nucleases (TALENS), 117 zinc-finger nucleases (ZFNS), 117 libraries, 232, 233

modification, 118, 119 structural modification, 74 Geocoris punctipes, 37

Geographical Indications Act, 174

Geranylgeranyl diphosphate (GGPP), 77

Germline nucleus, 129

Germplasm, 112

Global

climate change, 10, 120

warming, 84, 216

267

Glomerular stomach erosions, 194

Glycerol, 236

Glycine betaine biosynthesis, 116

Glycoalkaloids, 187

Glycoproteins, 114

Glycoside hydrolase enzymes, 113 Glyphosate, 28, 188–190, 193, 210, 212, 213

herbicides, 188

tolerant soybeans, 193

Glyphosphate, 77, 78, 191

resistant soyabeans, 78

Gossypium hirsutum L, 39

Government

health control authorities, 191

organizations, 179 policies, 250

regulatory bodies, 24

Gram-negative bacteria, 61

Granulocyte

colony-stimulating factors, 173, 198 macrophage colony-stimulating factors

(GCSF), 173

Green

fluorescence protein, 83 house effect, 84 gas emissions, 35

Growth

hormones, 134

promoters, 90, 246

Gut

epithelial cells, 32

receptors, 41

H Haemophilus, 225, 226, 228

aegyptius, 225

Hazard commercialization, 13 heavy metal-contaminated soils, 186

identification, 5 Health-related medicines, 164

Heat-stable studies, 57

Heavy metals, 90, 115, 139, 151

Helicoverpa

armigera, 25, 33, 36, 40, 92, 115, 137

zea, 38

Index

268 Heliothis

virescens, 209

zea, 92

Hematopoietic

stem cell, 139

system, 57

Hemophilia, 138, 223

Hepatitis B virus, 3, 95

Hepatocyte cells, 195

Herbal

reduction, 149

stipulations, 151

Herbicide, 26, 28, 29, 42, 58, 59, 77, 78,

85, 91, 92, 101, 117, 134, 137, 188–190,

208, 210, 212, 213, 246

applications, 188

resistance, 42, 85

crops (HRC), 137

tobacco, 78

tolerance, 26, 28, 58, 77, 117, 190

enzyme, 26 GM crops, 58, 190

plants, 26

Herbivore animals, 59

Hereditary emphysema, 85

Heterologous expression systems, 242

Heterozygous nature, 110 High

multiplication rate, 137

throughput DNA sequencing, 145

yielding varieties, 24

His3, 241

Homeostasis, 3, 14

Homologous recombination, 130, 235

Homopolymer tails, 234

Horticultural, 107, 108

grown plants, 107

plants, 107, 120

practices, 118

Host

expression vector system, 242, 243

genome, 79, 131

HrpN gene, 114

Human

albumin, 84

colon cancer cells proliferation, 120 consumption, 40, 41, 94, 137, 221

development hormones, 84

food chains, 194 genetic diseases, 138

growth hormone, 11, 95

immunodeficiency virus (HIV), 130 ingenuity, 171

insulin, 11, 12, 134, 174, 222

protein, 11

intervention, 2

microbiome, 191, 198

resource

development, 8

management, 7

sperm flagella, 129 therapeutic proteins, 119

viruses, 192

Humic substances, 188

Humulin, 12, 26

Hydrocarbons, 151, 166

Hypercholesterolemia, 13

Hypersensitivity, 114

I

Immature

red blood cells, 196

sperm production, 99

Immunological responses, 193

In vitro assessment, 56

methods, 112

Incidental mutation, 1

Incubation, 114, 234, 237

Indian

cotton industry, 42

Patent Office, 179 Indigenous microorganisms, 151

Inducible promoter genes, 254

Industrial

applicability, 160

fermentation process, 135 patents, 161

pollution, 151

products, 18

research, 134

Influenza virus, 130 Information sharing data management, 8

Index

269

Inorganic wastes, 95

Innovation authenticity, 167

Insect

infestation, 83 pest

abundance, 28

resistance, 42

resistance, 26, 85, 115

GM crop development, 59

Insecticidal, 28, 35, 36, 58, 59, 61, 77,

134, 137, 140, 189, 190

consumption, 37, 39

sprays, 37

toxin, 77

Institutional collaboration, 8

Integrated pest management, 37

Intellectual

property

organization (IPO), 179 protection (IPP), 1, 2, 18, 19, 21

rights (IPRs), 1, 17, 21, 76, 159, 173,

176, 177, 198

Intergeneric hybridization, 206 International

administrating bodies, 6

Agency for Research on Cancer (IARC), 191

Center for Genetic Engineering Biotechnology (ICGEB), 9

Depositary Authorities (IDA), 19

Plant Protection Convention (IPPC), 8

regulatory, 90

Interspecies hybridization, 125 Intragenesis, 207, 215

Inventions private ownership, 167

In-vitro fertilization, 130 Isoflavone, 94

J Japan Patent Office (JPO), 170, 179 Judicious breeding procedures, 75 Juvenile hormone (JH), 40

K Kanamycin, 25, 55

Keiferia lycorersicella, 92

Klenow DNA polymerase, 233

Korean Intellectual Property Office (KIPO), 179

Kurstaki, 25

L Labor-intensive techniques, 42

Lateral gene transfer, 187 Lectin induced proliferative growth, 194 Leghemoglobin, 146

Lepidoptera, 26, 30, 31, 190

borer complex, 35

pests, 33, 37

species, 24

Leptinotarsa decemlineata, 31, 115

Lethal microbes, 186

Leu2, 241

Ligase, 112, 170

chain reaction, 230

Lipolex, 239

Liposome, 133

lipoplex method, 238

mediated gene transfer, 79 Long coding RNA (lncRNA), 39

Local governmental bodies, 90

Low-cost improvement mechanisms, 42

Lutein, 113

Lytic peptides, 114

M Macronucleus, 129

Macrosiphum euphorbiae, 115

Maharashtra hybrid seed company

(MAHYCO), 30 Malaria transmission, 139

Malnourishment, 210

Malnutrition, 94, 210

problem, 28

Mammalian

cells, 138, 191, 237

expression system, 242

Map cloning, 80

Marker genes, 80

Market

agencies, 175

distribution, 58

Index

270 industrial transformation, 75 monopolies, 97, 248

Mass scale production, 175

Medicinal

fabrication, 84 properties, 134

Medico-legal complications, 170

Meganucleases, 131, 136

Meiosis process, 129

Meristematic tissues, 111

Mesenteric, 196

Metabolic

disorders, 99

endocrine drugs, 12

pathways, 191

Microbial

commercial activity notice, 62

communities, 188

genetics, 127

organisms patenting, 168

pesticides, 40

Microclimatic conditions, 190

Microinjection, 131, 134, 136 Microorganism, 17, 82, 84, 95, 125–127,

134, 140–146, 148, 149, 151, 152, 159,

164, 166–170, 173, 174, 178, 222

soil biota, 198

Midgut epithelium, 32

Migration genetic drift, 16 Mineralization, 149 Mitosis process, 129

Modern

agricultural set ups, 9

breeding techniques, 109

mutagenesis, 112

Modified tomatoes, 94 Molecular

approaches, 112

biology, 54, 73, 75, 76, 126, 129, 143,

146, 185

committee, 54

breeding, 187

markers, 112

mechanisms, 120

scissors, 81, 224, 225

Monarch butterfly, 23, 59, 100, 213 population, 100

Monoclonal antibodies, 84

Monocots, 189

Monopolies, 76, 97, 172

environment, 246

Monounsaturated oleic acid, 28

Mouse mammary tumor virus (MMTV), 231 Multicellular organisms, 128

Multinucleation, 194

Multiplicity reactivation, 130

Murine leukemia virus (MuLV), 231 Mutagenesis, 125, 145, 148, 191, 206, 207

agents, 110

process, 61

Mutational breeding, 108–110

Mycobacterium smegmatis, 127

Mycorrhiza, 28 Mycotic diseases, 208

Myllocerus spp., 36

N Nabis sp., 37

Naphthalene compounds degradation, 141

Narcissus, 77, 137

pseudo narcissus, 137

National Institute of Health (NIH), 11, 53,

54, 62, 63, 161

Native Cry protein activity, 31

Natural

biodiversity, 15, 16

ecosystem, 3, 5, 6, 9, 15, 16, 21, 119

gene pool, 59, 194

microbial community, 151

Necrosis, 194, 212

Nematocera, 25

Nematodes, 59

Neurospora

crasa, 128

fungi, 128, 129 Neurotransmitter, 13

Nezara viridula, 36

Nicotiana tobacum, 117

Non-functional gene product, 242 Non-GM myxoma virus-infected rabbit, 60 plants, 188

Nonhomologous end joining (NHEJ), 116,

117, 120

Index Nonrandom mating, 16

Nonsteroidal anti-inflammatory agents, 194 Non-target

crops, 100

entities, 76

insects, 56

organisms, 27, 59, 213, 247, 250–252,

254

Non-transgenic, 16, 36

Novel

organisms, 74

pathogenic varieties, 11

production processes, 148

scientific research, 151 Nucleases, 224

Nucleic acid, 10, 116, 129, 132, 146, 147,

164, 207, 224

editing tools, 116

Nucleotide

complementary, 231

sequence, 10, 81, 172, 224, 225

Nutrition

deficiencies, 152 disorders, 99

imbalance, 194

value, 23

O Obesity, 134

Octane compounds, 141

Oilseed, 119

Oligonucleotides, 230

Oligosaccharides, 144, 146, 148

Oocystis spp., 85

Organ transplantation, 15

Organization for Economic Co-Operation Development (OECD), 9, 41

Orius tristicolor, 37

Ornamental plant, 79, 93, 110

Orthodox

approaches, 209, 215

breeding techniques, 75

Osmotin, 208

Oxalate decarboxylase, 80

Oxucarenus laetus, 36

P

271

Palindrome

repeating sequence, 147

sequences, 226

Pancreatic acinar cells, 195

Papaya ringspot, 114

Paramecium species, 129

studies, 129

tetraurella, 129

Parasitic diseases, 138

Parasitization, 36 Paris International Convention for Protection of Birds (PICPB), 19 Patent, 160, 173

animals, 169

cooperation treaty (PCT), 19 gene transfer methods, 194 granting agencies, 176

holder rights, 172

living organisms, 159

office conduct examinations, 180 registration, 2

Pathogenic, 146

attacks, 94, 111

gut bacteria, 192

nucleic acid, 118

virus, 130

Pectinophora gossypiella, 25, 33, 137

Penicillin, 168

Peritrophic membrane, 32

Pest

infestation resistant varieties, 208 management, 26, 27, 42, 189

resistant traits, 209

resurgence, 24, 27

Pesticidal, 27, 34, 35, 37, 40, 91, 101, 189,

209, 246

consumption, 37

spry, 33

Petunia cells, 210

Phage capsid, 232

Pharmaceutical, 60, 84, 95, 138, 184

drugs, 55

industries, 12, 64

product, 15, 138

production, 138

Index

272 Pharmacological, 53

applications, 119

Phenol-chloroform process, 132 Phenotypic

characters, 108

effect, 130 traits, 109, 145

Phenylketonuria, 85

Phosphinotricin, 26

acetyltransferase, 26 Phosphorus excretion, 195

Photographs, 159, 175

Physical abrasion, 238

Phytochemicals, 119

Phytoene

desaturase, 77, 78, 94, 138

synthase

enzyme, 138 gene, 113

Pink bollworm (PBW), 25, 33, 38, 39, 137

Plant

biotechnology, 16

breeding, 75, 112, 116, 205, 209, 215

methodology, 112

techniques, 205

diets metabolism, 194

genome analyzation, 116 oriented vaccines, 95

Patent Act, 18

Protection Act, 169

varieties, 18, 58, 167, 209

Plasmid, 54, 61, 73–75, 77, 82, 127, 141,

222, 234, 241

efficiency, 82 Plasmodium, 60, 139

Polluted biotopes, 151

Polyamines, 238

Polycationic carbohydrates, 238

Polygalacturonase, 93

enzyme, 211 Polyhydroxyalkanoates (PHA), 139

Polymerase chain reaction (PCR), 78,

80–82, 131, 133, 136, 152, 180, 192,

193, 207, 216, 231

Polymeric compound, 237

Polynucleotide

chain, 224

kinase, 82

Polyplexes method, 238

Polyploids, 110

breeding, 110

Polyribosomes, 233

Poplar, 26, 206

Popularization, 42 Population explosion, 101, 102

Post

harvest operations, 208

translational modification, 112, 242 Predator-prey systems, 189

Premature births, 99

Production, 29, 33, 92, 101, 107

food substances, 140 human vaccines, 134

organisms, 147, 148

Products (RDT), 11 bovine growth hormone, 12

human growth hormone, 12

recombinant

insulin, 11

tryptophan, 13

Prokaryotic

cell, 237

expression systems, 242

organisms, 127

Promoter genes, 98, 99, 255

Protease, 146

Protection

biodiversity, 6, 8

property rights, 1

Protein

engineering, 147

eukaryotic expression system, 242

expression system, 242

macromolecules, 41

Proteolytic enzymes, 32 Protozoa, 59, 126, 129 species, 129

Pro-vitamin A, 94

Pseudomonas, 60, 61, 150

Publication, 159, 162

experimentation, 179

Q Quality products, 89

Index

R

273

disease, 194

pathogenic strains, 74, 85

Ralstonia eutropha, 139

Resource

Rapid generations advances (RGA), 109

accessibility, 189

Recombinant cry1Ab gene fragments, 193 depletion, 119

Recombinant

Restriction

DNA (rDNA), 11, 12, 18, 53–55, 57,

endonucleases (DNA cutting enzymes), 59–64, 74–76, 78, 79, 82, 82, 86, 130,

81, 170, 207, 224, 225, 227

131, 133, 136, 139, 141, 146, 150,

nomenclature, 225

183–185, 197, 206, 207, 222–224,

recognition sequences, 226

230–232, 234, 240

uses of restriction enzymes (RES), 229 technology, 10, 76, 79, 184, 206, 207,

enzymes (REs), 81, 82, 112, 117, 131,

222

133, 180, 184, 224, 225, 227–229,

environment,

234, 241

harmful effect of, 185 fragment length polymorphism (RFLP), antibiotic resistant genes, 192

80, 229

effects of GM food on gastrointestinal modification system, 225 tract, 194

nucleases, 232

effects of GM food on pancreas, 195 Retrovirus, 82, 134

effects of RDNA technology (biodiversity), 189

Reverse

effects of RDNA technology (soil), genetics, 132

188

transcriptase, 230

food allergenicity testing, 196 Rhabdovirus, 4

harmful effect of RDNA (human

Rhizomes, 16 health), 190

Rhizosphere, 61, 142 harmful impact of RDNA technology Rhodococcus, 150

(animals), 194

Ribonuclease-H, 231

impact on human health, 191

Ricin, 187

modifications (hematology), 195 RNA dependent DNA synthetase, 230

possible assimilation of genes, 193 Rod-shaped bacterium, 231

hepatitis B vaccine, 138

Root exudates, 188

microorganisms, 61

Rous sarcoma virus (RSV), 231 molecules, 54

construction, 223

S organism, 61, 62, 232, 234

Salicylate, 141

progeny, 130

Saline environments, 208

proteins, 84

Salmonella, 77, 239

strains (organisms), 84

Salt

strands, 82

concentration, 147

technology (RDT), 10–12, 13, 25, 28,

resistance, 85

54, 58, 59, 63, 64, 76, 79, 80, 130,

tolerance, 116, 208

131, 136, 141, 183–185, 197, 206,

Scientific 222, 232

community, 6, 12, 23

manufactured tryptophan, 13 organizations, 179 Renal lysozyme levels, 195 Screening

Resistance

process, 132, 133

development, 37, 38

Index

274 recombinant clone, 239

antibiotic sensitivity, 241

chromogenic substrate, 239

complementation (mutation), 241

insertional inactivation (antibiotic

resistance gene), 240

insertional inactivation method, 240

Secondary metabolites, 36, 118–120

Secure isotope probing (SIP), 150, 180

Seed-specific protein, 80 Selectable

breeding, 205, 206, 210

markers, 136

Self immune crops, 42

pollination, 254

replication, 82

reported lifetime allergic responses, 196 Septicemia, 32

Sequence, 73, 74, 114, 131, 136, 164, 170,

229

specificity, 226 techniques, 131

Serine proteases, 39

Serotonin, 13

Serratia marcescens, 227

Severe acute respiratory syndrome corona-

virus (SARS-CoV-2), 162, 198 Shigella, 239

Shrinking cultivable land, 42

Sickle cell

anemia, 139

hemoglobin disease, 117

Site-specific nucleases (SSNs), 207, 243 Small interfering RNAs (siRNAs), 39 Socioeconomic wellbeing, 167

Sodium, 92, 195

Soil

erosion, 76, 189

micro-environment, 187

salinity, 40

Solanum demissum, 111

Solar UV-rays, 24 Somatotrophin, 12

Sotto disease, 24

Source organism, 242

Specific foreign nucleic acid bacteria, 25

Speed breeding, 109, 112, 113

Spermosphere, 61

Spider silk polymers, 95

Spodoptera

frugiperda, 25, 31, 212

litura, 25, 33, 36, 209

Stakeholder farmers, 33 Staphylococcus, 226, 229

Stationary-phase promoters, 150

Stem cell technology, 139

Steviol, 144, 146, 148

Stomach erosion, 194

Straight microinjection, 74 Streptococcus, 225

Streptomyces hygroscopicers, 26

Subtractive hybridization, 80 Sugar

cane, 26, 110, 206

composition, 36

Suicidal

farmers, 40 genetically engineered microorganisms

(S-GEMS), 150, 180

Summary information format for environment release (SIFER), 60

Superbugs, 141

Sustainability, 33, 212, 250

agriculture, 119

plant protection approaches, 24

Symbiotic associations, 189

Symptomatology, 212

Synthetic

chemicals, 24

functions, 222 oligonucleotides, 214

Systematic pyrethroids, 36

Systemic acquired resistance (SAR), 114, 120

T TAL effector (TALE), 214 Target genome modification, 116 lepidopteran pests, 31

Technology innovations, 168

intervention, 2

Terrestrial ecosystems, 190

Index Tetracycline containing medium, 241 gene, 240 resistance genes, 234 Tetranychus cinnabarinus, 37 Textile industry, 32 Therapeutic products, 84, 85 Thermal denaturation, 230 Thermus aquaticus, 225, 231 Thrips tabaci, 33, 36 Tissue culture

cells, 79

techniques, 55, 109, 111

plasminogen activator, 11

Tobacco plant, 55, 78 Tolerance mechanism, 209 Tomato leaf curl virus activity, 210 Top-of-line clean-up agent, 150 Totipotency, 131 cells, 170 Toxicity, 24, 26, 55–57, 64, 126, 150, 184, 187, 190, 192, 194, 198, 238

generating microbes, 55

Trade secrets, 159, 175, 176 Trademarks, 17, 18, 159, 160, 175, 177, 178 Traditional breeding, 108, 109

methods, 108

cross-fertilization techniques, 93 food developments, 144 pesticides, 41

vaccines, 95

Traits, 7, 25, 26, 28, 44, 74, 76, 77, 79, 83, 85, 86, 93, 97, 98, 101, 108–112, 118, 119, 128, 147, 171, 183, 186, 205, 211, 215, 221, 222, 240 Transcription activator, 116, 117, 120, 152, 243 like effector DNA binding site, 117 like effector nucleases (TALEN), 79, 116, 117, 131, 136, 137, 147, 152, 207, 214, 215, 243 mediated gene editing techniques, 214 Transduction, 82, 127, 133, 134, 239 Transfection, 81, 126, 133, 238, 239 Transformation, 28, 82, 133, 234

275

Transgenes, 21, 81, 186, 187, 189, 191, 206, 207, 254

transient expression, 191

Transgenesis, 136 Transgenic, 3, 10, 11, 15, 16, 18, 26, 27,

55, 56, 74, 77, 78, 80, 81, 83, 85, 90, 96, 98, 108, 111–114, 116, 119, 134, 135, 164, 169, 170, 187, 189, 192–195, 206–208, 210–213, 245, 247–249, 251, 254, 255 animals, 85, 134, 135, 169, 170 breeding method, 111 cells, 81, 83 corn, 77 crops, 56, 83, 85, 112, 192, 210, 213, 247 food, 16 genes fragments, 192 methods, 108 non-human mammals, 18 organism, 15, 55, 90, 96, 98, 119, 192, 245, 247–249, 254, 255 plant, 16, 26, 55, 77, 78, 80, 85, 113, 114, 119, 189, 192, 206, 210 products, 3 species, 15 varieties, 11, 15, 213 plants, 11 Transient expression (CRISPR Cas9), 118 Transmissible changed nourishments, 83 Transpiration levels, 92 Transplantation, 170 Transposon, 127 mediated gene transfer, 79 Troubled tri-trophic interactions, 189 Trypsin, 39, 115, 143, 144 Tryptophan, 11, 13 Tubular degenerative changes, 195 Tumor suppressing genes, 14 Tungsten, 133 Turnip mosaic virus, 114

U Ultraviolet radiation, 145 Unicellular organisms, 129 Uniform Trade Secrets Act (UTSA), 176 Union for Protection of New Varieties (UPOV), 18

Index

276 United

Kingdom (UK), 58, 97, 99, 189, 248

Nations Organization (UNO), 2, 18 States Department of Agriculture, 136 US Environmental Protection Agency

(US-EPA), 24, 78

US Patent Trademark Office (USPTO), 179

US supreme court proceedings-afterwards, 167

biotech industry, 168

comprehension (patents innovations), 167

courts decision influence, 167 patent law-PTO, 168 USA Department of Agriculture (USDA),

11, 54, 63, 136

V

Vaccination treatment, 4 Vaccine, 4, 60, 84, 85, 95, 101, 119, 138,

162, 164

production, 84

Vaccinia virus, 130

Vanillin, 144 Vegetative propagated, 111

crops, 109

reproductive bodies, 16

tissues, 108

Viral protein, 162

vector-mediated gene transfer, 79 Virus, 129, 134 protection, 26

resistant

squash, 78

varieties, 27

Vitamin A, 29, 111, 138 deficiency, 77 rich golden rice, 26

Vitamin, 101, 111, 144, 148, 149 rich golden rice, 28

Voltinism, 37

W Weather-resistant crops, 147

Weedicide resistant transgenic crop plants,

76

Western Hemisphere Convention (WHC),

19

Wildlife resources, 19 World Food Prize, 79 Health Organization, 198 Intellectual Property Organization, 18 International Protection Organization

(WIPO), 2, 18, 162

Trade Organization (WTO), 9, 160

X

Xanthomonas axonopodis, 114

Xenobiotic compound, 126, 142

Xeno-transplant, 15

X-gal-lacZ system, 83 Xylene compounds degradation, 141

Y Yogurt, 126, 143, 144

Z Zea mays, 117

Zelus renardii, 37

Zinc fingers (ZF), 214 nucleases (ZFNs), 116, 117, 120, 131,

136, 207, 214, 243

protein, 117

Zucchini yellow mosaic virus, 114